Compositions comprising tissue specific adenoviral vectors

ABSTRACT

Compositions comprising host cell specific adenovirus vehicles are provided for transfecting target host cells. The compositions comprise replication competent adenovirus having an adenovirus gene essential for replication under transcriptional control of a cell type specific transcriptional response element (TRE) and polyethylene glycol (PEG) as a masking agent.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/509,591, filed Jun. 2, 2000; and a continuation-in part ofapplication Ser. No. 09/151,376, filed Sep. 10, 1998, which is acontinuation-in-part of application Ser. No. 08/699,753, filed Jun. 26,1996, which is a continuation-in-part of application Ser. No.08/495,034, filed Jun. 27, 1995, the disclosure of which is hereinincorporated by reference. This application is also a continuation-inpart of application Ser. No. 09/033,428, filed Mar. 2, 1998, whichclaims the benefit of provisional application serial. No. 60/039,597,filed Mar. 3, 1997; and a continuation-in-part of application Ser. No.09/033,555, filed Mar. 2, 1998, which claims the benefit of provisionalapplication serial No. 60/039,763 filed Mar. 3, 1997; and acontinuation-in-part of application Ser. No. 09/033,333, filed Mar. 2,1998, which claims the benefit of provisional application serial No.60/039,762, filed Mar. 3, 1997. All of the above patent applications areincorporated by reference herein.

TECHNICAL FIELD

[0002] The field of this invention is cell transfection, particularly byadenoviral vectors.

BACKGROUND

[0003] The ability to change the genotype and phenotype of cells invitro and in vivo has many applications. For studying physiologicprocesses, particularly with dedicated cells, there is substantialinterest in being able to modify the phenotype to affect a particularprocess. By enhancing or depressing the amount of a member of thephysiological pathway, by inhibiting the activity of a member of thepathway, by providing an allele or mutated analog of the naturallyoccurring member, one may be able to unravel the role of the variousmembers in the pathway, the order in which the members participate, thepresence of alternative pathways and the like. Also, one can use thecells for producing proteins.

[0004] Adenovirus does not require cell proliferation for efficienttransduction of cells. Adenovirus modified by introduction of atransgene provides for transient expression of proteins. Adenovirus canbe rendered incompetent by inactivating one or more essential genes andthen be packaged in a helper cell line for use in transfection. Thus,adenovirus affords a convenient vehicle for modifying cellular traits orkilling cells, as appropriate.

[0005] For many medical applications, there is an interest in being ableto specifically modify target cells in vivo or ex vivo. The modificationcan be associated with random DNA integration, whereby a geneticcapability is introduced that complements a genetic defectintracellularly, provides for secretion of a product from the modifiedcells, which is otherwise undetectably produced or not produced by thehost, provide protection from disease, particularly viral disease, andthe like. In many situations, in order to be effective, one must have ahigh efficiency of transfection of the target cells. This isparticularly true for in vivo modification. In addition, one would wishto have a high specificity for the target cells, as compared to othercells that may be present ex vivo or in vivo.

[0006] Gene therapy involves the transfer of cloned genes to targetcells. A variety of viral and non-viral vehicles have been developed totransfer these genes. Of the viruses, retroviruses, herpes virus,adeno-associated virus, Sindbis virus, poxvirus and adenoviruses havebeen used for gene transfer. These vehicles all have differentproperties. For example, retroviruses transduce genes in vitro with highefficiency by integrating the transduced gene into the chromosomefollowing division of infected cells. Adeno-associated viruses canstably integrate into and express transduced genes in both dividing andquiescent cells. In contrast, liposomes and adenovirus allow onlytransient gene expression, and transduce both dividing and quiescenttarget cells.

[0007] Of the viruses, adenoviruses are among the most easily producedand purified, whereas retroviruses are unstable, difficult to produceand impossible to purify. Both classes of virus transduce cells withhigh efficiency. Liposomes hold the promise of allowing repeat doses ofgenes for, unlike viruses, they are not immunogenic. However, liposomescompleted with DNA are difficult to produce in commercial quantities,and are inefficient gene transfer vehicles, most often transducing fewerthan one percent of target cells.

[0008] Publications describing various aspects of adenovirus biologyand/or techniques relating to adenovirus include the following. Grahamand Van de Eb (1973) Virology 52:456-467; Takiff et al. (1981) Lancetii:832-834; Berkner and Sharp (1983) Nucleic Acid Research 6003-6020;Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J. Virology67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806 describe adenoviruses that have been genetically modifiedto produce replication-defective gene transfer vehicles. In thesevehicles, the early adenovirus gene products E1A and E1B are deleted andprovided in trans by the packaging cell line 293 developed by FrankGraham (Graham et al. (1987) J. Gen. Birol. 36:59-72 and Graham (1977)J. Genetic Virology 68:937-940). The gene to be transduced is commonlyinserted into adenovirus in the deleted E1A and E1B region of the virusgenome Bett et al. (1994), supra. Adenovirus vectors as vehicles forefficient transduction of genes have been described byStratford-Perricaudet (1990) Human Gene Therapy 1:2-256; Rosenfeld(1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol.309:61-66; Jaffe et al. (1992) Nat Gent. 1:372-378; Quantin et al.(1992) Proc Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992)Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest.90:626-630; Le Gal La Salle et al. (1993) Science 259:988-990;Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al.(1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem.269:23872-23875.

[0009] There are two major divisions of gene therapy protocols: in vivoand ex vivo. In vivo refers to administration of the therapeuticdirectly to the patient, usually by inhalation or injection, althoughoral administration has been suggested in some instances. Ex vivo genetherapy refers to the process of removing cells from a patient, forexample in a biopsy, placing the cells into tissue culture, transferringgenes to the cells in tissue culture, characterizing the newlygenetically engineered cells, and finally returning the cells to thepatient by intravenous infusion. Therapeutically, retroviruses are mostoften used for ex vivo transfer, whereas adenoviruses and liposomes aremost often used for in vivo gene transfer.

[0010] In the treatment of cancer by replication-defective adenoviruses,the host immune response limits the duration of repeat doses of thetherapeutic at two levels.. First, the adenovirus delivery vehicleitself is immunogenic. Second, late virus genes are frequently expressedin transduced cells, eliciting cellular immunity. Thus, the ability torepeatedly administer cytokines, tumor suppressor genes, ribozymes orsuicide genes is limited by the transient nature of gene expression, andthe immunogenicity of both the gene transfer vehicle and the viral geneproducts of the transfer vehicle.

[0011] There is, therefore, substantial interest in being able todevelop viral vectors which substantially reduce the present limitationsand restrictions on the use of such vectors in vivo.

SUMMARY OF THE INVENTION

[0012] Replication-competent adenovirus vectors, and methods for theiruse as vehicles for the transduction of restricted cell types, areprovided. The invention provides compositions comprising replicationcompetent adenovirus having an adenovirus gene essential for replicationunder transcriptional control of a cell type specific transcriptionalresponse element (TRE), and polyethylene glycol (PEG) as a maskingagent.

[0013] In one aspect, this adenoviral gene is an early gene.Additionally, one or more late genes and/or one or more transgenes maybe under the control of a transcriptional initiation region that istranscriptionally active only in the target cells of interest. In someembodiments, the replication competent adenovirus comprises a transgene.

[0014] The cell type specific TRE may be prostate cell specific and mayinclude the promoter sequence found at nucleotides −540 to +8 relativeto transcription start site of prostate specific antigen gene and/or theenhancer sequence in the region −5322 to −3739 relative to thetranscription start site of the prostate specific antigen gene.

[0015] In other embodiments, the prostate cell specific response elementis a probasin TRE or a hKLK2-TRE.

[0016] In still other embodiments, an adenovirus gene essential forreplication is under transcriptional control of an α-fetoproteintranscriptional regulatory element (AFP-TRE), a MUC1-TRE or a CEA-TRE.

[0017] The composition may include PEG of a molecular weight betweenabout 2500 to about 30,000, 3000 to about 20,000, or 5000 to about10,000 and the PEG may be covalently or non-covalently attached to theadenovirus.

[0018] In embodiments where the PEG is covalently attached to theadenovirus, the attachment may be by way of an N-hydroxysuccinimidyl(NHS) active ester such as succinimidyl succinate, succinimidylsuccinamide or succinimidyl propionate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a schematic representation of various adenoviral vectorconstructs comprising AFP-TRE controlling expression of E1A, E1B orboth, as described in Example 3.

[0020] FIGS. 2(A) and (B) are half tone reproductions depicting westernanalysis of E1A levels in CN733 (containing two AFP-TREs) and CN702(control) infected cells. In FIG. 2(A), the left panel shows Huh-7(AFP+) cells; the right panel shows DId-1 (AFP−) cells. In FIG. 2(B),Sk-Hep-1 were the AFP− cells used.

[0021] FIGS. 3(A)-(C) are graphs depicting growth of CN733 in AFPproducing (Huh-7; FIG. 3(A)) and non-AFP producing (Sk-Hep-1, FIG. 3(B);DId-1, FIG. 3(C)) cells.

[0022] FIGS. 4(A)-(C) are graphs depicting growth of CN732 (FIG. 4(A);solid diamonds), CN733 (FIG. 4(B); solid diamonds), and CN734 (FIG.4(C); solid diamonds) in HepG2 cells, as compared to control CN702(solid squares).

[0023] FIGS. 5(A)-(B) are graphs depicting growth of CN732 (FIG. 5(A);solid squares), and CN733 (FIG. 5(B); solid circles) in primaryhepatocytes, compared to control CN702 (solid diamonds).

[0024] FIGS. 6(A)-(B) are graphs comparing tumor volume in miceharboring hepatocarcinoma cell line HepG2 and treated with CN733 (FIG.5(A); squares) or with control buffer (circles). FIG. 6(A) depictsmeasuring tumor volume over a period of 43 days (six weeks). In FIG.6(B), single intratumoral administration of CN733 (“B”) was compared tofive consecutive daily doses of CN733 (“J”).

[0025]FIG. 7 is a graph depicting serum AFP levels in tumor-bearing micereceiving CN733 (triangles) or receiving buffer (circles).

[0026]FIG. 8 is a schematic representation of various adenoviral vectorconstructs comprising CEA-TRE controlling expression of E1A, E1B orboth, as described in Example 4.

[0027]FIG. 9 is a schematic representation of various adenoviral vectorconstructs comprising MUC-TRE controlling expression of E1A, E1B orboth, as described in Example 5.

[0028]FIG. 10 (SEQ ID NO: 9) depicts the sequence of the 5′-flankingregion of the rat probasin (PB) gene, including the PB-TRE region.Numbers above the nucleotides indicate position relative to thetranscription start site. The locations of androgen response elements(ARE) are indicated.

[0029]FIG. 11 depicts schematic diagrams of various adenovirus vectorsin which various genes are under control of a PB-TRE.

[0030]FIG. 12 is a schematic representation of the hKLK2-TREs usedto-generate the adenoviral constructs described in Example 7.

[0031]FIG. 13 is a schematic representation of the adenoviral constructsdescribed in Example 7, in which adenoviral genes E1A and E1B are undertranscriptional control of hKLK2-TREs. The ovals indicate that theendogenous E1A is present. The triangles indicate that the endogenousE1B promoter was removed. Abbreviations for TREs are as follows: hKLK2P: hKLK2 promoter; hKLK2 (1.8 E+P): 1.8 kb hKLK2 enhancer and minimalhKLK2 promoter, as depicted in FIG. 12; hKLK2 (1.17 kb E+P): 1.17 kbhKLK2 enhancer and minimal hKLK2 promoter, as depicted in FIG. 12.

[0032]FIG. 14 depicts a nucleotide sequence of a carcinoembryonicantigen TRE.

[0033]FIG. 15 depicts a nucleotide sequence of a prostate-specificantigen TRE.

[0034]FIG. 16 depicts a nucleotide sequence of a human glandularkallikrein TRE.

[0035]FIG. 17 depicts a nucleotide sequence of a mucin TRE.

[0036]FIG. 18 depicts a nucleotide sequence of a rat probasin TRE.

[0037]FIG. 19 depicts a nucleotide sequence and translated amino acidsequence of an adenovirus death protein.

[0038]FIG. 20 is a schematic depiction of a method for covalentpegylation of an adenovirus. In this method, succinimidyl succinamide isused to covalently attach methoxy-PEG to adenovirus. The pegylatedadenovirus is separated from the reaction components by ion exchangechromatography.

[0039]FIG. 21 shows a half-tone reproduction depicting a sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (mobilityshift) of pegylated adenovirus proteins. Lanes 1 and 2 are non-pegylatedCN706 (control), lanes 3 through 6 are CN706 pegylated under various pHand temperature conditions (lane 3, pH 7.6, room temperature (RT); lane4, pH 7.6, 4° C.; lane 5, pH 8.2, RT, lane 6, pH 8.2, 4° C.).

[0040]FIG. 22 is a chromatogram of ion exchange chromatography analysisof pegylated adenovirus (PEG-706) mixed with control adenovirus CN706.

MODES FOR CARRYING OUT THE INVENTION

[0041] The present invention is based on the discovery and constructionof replication-competent adenovirus vectors containing celltype-specific transcriptional regulatory elements (TREs) which canpreferentially replicate in cells that allow function of said TREs, andmethods of using these adenovirus vectors. The adenovirus vectors ofthis invention comprise an adenovirus gene under the transcriptionalcontrol of a cell type-specific TRE. Preferably, the adenovirus gene isone that enhances, i.e. promotes, cell death, more preferably one thatis essential for adenovirus replication. Preferably, the adenovirus genenecessary for cell replication is an early gene. In some embodiments,the adenovirus vectors of this invention comprise an adenovirus geneunder the transcriptional control of a cell type-specific TRE, and atleast one other gene, such as an adenoviral gene or a transgene, undercontrol of a second cell type-specific TRE which is substantiallyidentical to the first TRE. Preferably, the first and second genes undertranscriptional control of the cell type-specific TREs are bothadenovirus genes necessary for replication. By providing forcell-specific transcription through the use of one or more celltype-specific TREs, the invention provides adenovirus vectors that canbe used for cell-specific cytotoxic effects due to selectivereplication.

[0042] The adenovirus vectors of the invention replicate preferentiallyin TRE functional cells (i.e., at a higher yield than in TREnon-functional cells), referred to herein as target cells. Thisreplication preference is indicated by comparing the level ofreplication (i.e., titer) in cells in which the TRE is active to thelevel of replication in cells in which the TRE is not active (i.e., anon-target cell). The replication preference is even more significant,as the adenovirus vectors of the invention actually replicate at asignificantly lower rate in TRE non-functional cells than wild typevirus. Comparison of the adenovirus titer of a target cell to the titerof a TRE inactive cell type provides a key indication that the overallreplication preference is enhanced due to the replication in targetcells as well as depressed replication in non-target cells. This isespecially useful in the cancer context, in which targeted cell killingis desirable.

[0043] Runaway infection is prevented due to the cell-specificrequirements for viral replication and to this instability, which may bemediated by recombination between TREs. Without wishing to be bound byany particular theory, the inventors note that production of adenovirusproteins can serve to activate and/or stimulate the immune system,either generally or specifically toward target cells producingadenoviral proteins which can be an important consideration in thecancer context, where patients are often moderately to severelyimmunocompromised.

[0044] Adenovirus vectors have been constructed in which each of the E1Aand E1B genes have been placed under transcriptional control of celltype-specific TREs, for example, TREs from the PSA gene (PSE-TRE), theprobasin gene (PB-TRE), the hKLK2 gene (hKLK2-TRE), the a-fetoproteingene (AFP-TRE), the carcinoembryonic antigen gene (CEA-TRE), and themucin gene (MUC-TRE).

[0045] The adenovirus vectors, in which cell type-specific TREs are usedto control replication, achieve a high level of target cell specificity.

[0046] General Techniques

[0047] The practice of the present invention will employ, unlessotherwise, indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the, literature, such as, “MolecularCloning: A Laboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991).

[0048] For techniques related to adenovirus, see, inter alia, Felgnerand Ringold (1989) Nature 337:387-388; Berkner and Sharp (1983) Nucl.Acids Res. 11:6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al.(1993) J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl. Acad.Sci. USA 91:8802-8806.

[0049] Definitions

[0050] An “adenovirus vector” or “adenoviral vector” (usedinterchangeably) is a term well understood in the art and generallycomprises a polynucleotide (defined herein) comprising all or a portionof an adenovirus genome. As used herein, “adenovirus” refers to thevirus itself or derivatives thereof. The term covers all serotypes andsubtypes and both naturally occurring and recombinant forms, exceptwhere otherwise indicated. For the purposes of the present invention, anadenovirus vector contains a cell type-specific TRE operably linked toan adenovirus gene, and may optionally contain a second adenoviral geneor a transgene operably linked to a cell type-specific TRE or anothertype of TRE, which is non-cell type-specific. An adenoviral vector ofthe present invention can be in any of several forms, including, but notlimited to, naked DNA; an adenoviral vector encapsulated in anadenovirus coat; packaged in another viral or viral-like form (such asherpes simplex virus and AAV); encapsulated in a liposome; complexedwith polylysine or other biocompatible polymer; complexed with syntheticpolycationic molecules; conjugated with transferrin; complexed withcompounds such as PEG to immunologically “mask” the molecule and/orincrease half-life, or conjugated to a non-viral protein. An adenoviralvector of this invention maybe in the form of any of thedelivery-vehicles described herein. Such vectors are one embodiment ofthe invention. Preferably, the polynucleotide is DNA. As used herein,“DNA” includes not only bases A, T, C, and G, but also includes any oftheir analogs or modified forms of these bases, such as methylatednucleotides, internucleotide modifications such as uncharged linkagesand thioates, use of sugar analogs, and modified and/or alternativebackbone structures, such as polyamides. For purposes of this invention,adenovirus vectors are replication-competent in a target cell.

[0051] As used herein, a “transcriptional regulatory element”, or “TRE”is a polynucleotide sequence, preferably a DNA sequence, that regulates(i.e., controls) transcription of an operably-linked polynucleotidesequence by an RNA polymerase to form RNA. As used herein, a TREincreases transcription of an operably linked polynucleotide sequence ina host cell that allows the TRE to function. The TRE comprises anenhancer element and/or promoter element, which may or may not bederived from the same gene. The promoter and enhancer components of aTRE may be in any orientation and/or distance from the coding sequenceof interest, and comprise multimers of the foregoing, as long as thedesired transcriptional activity is obtained. As discussed herein, a TREmay or may not lack a silencer element.

[0052] An “enhancer” is a term well understood in the art and is apolynucleotide sequence derived from a gene which increasestranscription of a gene which is operably-linked to a promoter to anextent which is greater than the transcription activation effected bythe promoter itself when operably-linked to the gene, i.e. it increasestranscription from the promoter. Having “enhancer activity” is a termwell understood in the art and means what has been stated, i.e., itincreases transcription of a gene which is operably linked to a promoterto an extent which is greater than the increase in transcriptioneffected by the promoter itself when operably linked to the gene, i.e.,it increases transcription from the promoter.

[0053] “Under transcriptional control” is a term well-understood in theart and indicates that transcription of a polynucleotide sequence,usually a DNA sequence, depends on its being operably (operatively)linked to an element which contributes to the regulation of, eitherpromotes or inhibits, transcription.

[0054] The term “operably linked” relates to the orientation ofpolynucleotide elements in a functional relationship. A TRE is operablylinked to a coding segment if the TRE promotes transcription of thecoding sequence. Operably linked means that the DNA sequences beinglinked are generally contiguous and, where necessary to join two proteincoding regions, contiguous and in the same reading frame. However, sinceenhancers generally function when separated from the promoter, byseveral kilobases and intronic sequences may be of variable length, somepolynucleotide elements may be operably linked but not contiguous.

[0055] A “cell type-specific TRE” is preferentially functional in aspecific type of cell relative to other types of cells of differentfunctionality. “Cell type” is a reflection of a differentiation state ofa cell which is, under normal physiological conditions, an irreversible,end-stage state. For example, a prostate-specific antigen TRE isfunctional in prostate cells, but is not substantially functional inother cell types such as hepatocytes, astrocytes, cardiocytes,lymphocytes, etc. Generally, a cell type-specific TRE is active in onlyone cell type. When a cell type-specific TRE is active in more than onecell type, its activity is restricted to a limited number of cell types,i.e., it is not active in all cell types. A cell type-specific TRE mayor may not be tumor cell specific.

[0056] As used herein, the term “cell type-specific” is intended to meanthat the TRE sequences to which a gene, which may be a gene essentialfor replication of an adenoviral vector, is operably linked, or to whicha transgene is operably linked, functions specifically in that targetcell so that transcription (and replication, if the operably linked geneis one essential for adenovirus replication) proceeds in that targetcell, or so that a transgene polynucleotide is expressed in that targetcell. This can occur by virtue of the presence in that target cell, andnot in non-target cells, of transcription factors that activatetranscription driven by the operably linked transcriptional controlsequences. It can also occur by virtue of the absence of transcriptioninhibiting factors that normally occur in non-target cells and preventtranscription driven by the operably linked transcriptional controlsequences. The term “cell type-specific”, as used herein, is intended toinclude cell type specificity, tissue specificity, as well asspecificity for a cancerous state of a given target cell. In the lattercase, specificity for a cancerous state of a normal cell is incomparison to a normal, non-cancerous counterpart.

[0057] As used herein, a TRE derived from a specific gene is referred toby the gene from which it was derived and is a polynucleotide sequencewhich regulates transcription of an operably linked polynucleotidesequence in a host cell that expresses said gene. For example, as usedherein, a “human glandular kallikrein transcriptional regulatoryelement”, or “hKLK2-TRE” is a polynucleotide sequence, preferably a DNAsequence, which increases transcription of an operably linkedpolynucleotide sequence in a host cell that allows an hKLK2-TRE tofunction, such as a cell (preferably a mammalian cell, even morepreferably a human cell) that expresses androgen receptor. An hKLK2-TREis thus responsive to the binding of androgen receptor and comprises atleast a portion of an hKLK2 promoter and/or an hKLK2 enhancer (i.e., theARE or androgen receptor binding site).

[0058] As used herein, a.“probasin (PB),transcriptional regulatoryelement”, or“PB-TRE” is a polynucleotide sequence, preferably a DNAsequence, which selectively increases transcription of anoperably-linked polynucleotide sequence in a host cell that allows aPB-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses androgen receptor. A PB-TREis thus responsive to the binding of androgen receptor and comprises atleast a portion of a PB promoter and/or a PB enhancer (i.e., the ARE orandrogen receptor binding site).

[0059] As used herein, a “prostate-specific antigen (PSA)transcriptional regulatory element”, or “PSA-TRE”, or “PSE-TRE” is apolynucleotide sequence, preferably a DNA sequence, which selectivelyincreases transcription of an operably linked polynucleotide sequence ina host cell that allows a PSA-TRE to function, such as a cell(preferably a mammalian cell, even more preferably a human cell) thatexpresses androgen receptor. A PSE-TRE is thus responsive to the bindingof androgen receptor and comprises at least a portion of a PSA promoterand/or a PSA enhancer (i.e., the ARE or androgen receptor binding site).

[0060] As used herein, a “carcinoembryonic antigen (CEA) transcriptionalregulatory element”, or “CEA-TRE” is polynucleotide sequence, preferablya DNA sequence, which selectively increases transcription of an operablylinked polynucleotide sequence in a host cell that allows a CEA-TRE tofunction, such as a cell (preferably a mammalian cell, even morepreferably a human cell) that expresses CEA. The CEA-TRE is responsiveto transcription factors and/or co-factor(s) associated withCEA-producing cells and comprises at least a portion of the CEA promoterand/or enhancer.

[0061] As used herein, an “α-fetoprotein (AFP) transcriptionalregulatory element”, or “AFP-TRE” is polynucleotide sequence, preferablya DNA sequence, which selectively increases transcription (of anoperably linked polynucleotide sequence) in a host cell that allows anAFP-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses AFP. The AFP-TRE isresponsive to transcription factors and/or co-factor(s) associated withAFP-producing cells and comprises at least a portion of the AFP promoterand/or enhancer.

[0062] As used herein, an “a mucin gene (MUC) transcriptional regulatoryelement”, or “MUC1-TRE” is a polynucleotide sequence, preferably a DNAsequence, which selectively increases transcription (of anoperably-linked polynucleotide sequence) in a host cell that allows anMUC1-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses MUC1. The MUC1-TRE isresponsive to transcription factors and/or co-factor(s) associated withMUC1-producing cells and comprises at least a portion of the MUC1promoter and/or enhancer.

[0063] As used herein, a “target cell” is one which allows (i.e.,permits or induces) a cell type-specific TRE to function. Preferably, atarget cell is a mammalian cell, preferably a human cell.

[0064] As used herein, “a cell which allows a TRE to function” or a cellin which the function of a TRE is “sufficiently preserved” or“functionally preserved”, or “a cell in which a TRE is functional” is acell in which the TRE, when operably linked to a promoter (if notincluded in the TRE) and a reporter gene, increases expression of thereporter gene at least about 2-fold, preferably at least about 5-fold,preferably at least about 10-fold, more preferably at least about20-fold, more preferably at least about 50-fold, more preferably atleast about 100-fold, more preferably at least about 200-fold, even morepreferably at least about 400-to about 500-fold, even more preferably atleast about 1000-fold, when compared to the expression of the samepromoter and reporter gene when not operably linked to said TRE. Methodsfor measuring levels (whether relative or absolute) of expression areknown in the art and are described herein.

[0065] The activity of a TRE generally depends upon the presence oftranscriptional regulatory factors and/or the absence of transcriptionalregulatory inhibitors. Transcriptional activation can be measured in anumber of ways known in the art (and described in more detail below),but is generally measured by detection and/or quantitation of mRNA orthe protein product of the coding sequence under control of (i.e.,operatively linked to) the TRE. As discussed herein, a TRE can be ofvarying lengths, and of varying sequence composition. By transcriptionalactivation, it is intended that transcription will be increased abovebasal levels in the target cell by at least about 2-fold, preferably atleast about 5-fold, preferably at least about 10-fold, more preferablyat least about 20-fold. More preferably at least about 50-fold, morepreferably at least about 100-fold, even more preferably at least about200-fold, even more preferably at least about 400- to about 500-fold,even more preferably, at least about 1000-fold. Basal levels aregenerally the level of activity, if any, in a non-target cells, or thelevel of activity (if any) of a reporter construct lacking the TRE ofinterest as tested in a target cell type.

[0066] A “functionally-preserved” variant of a TRE is a TRE whichdiffers from another TRE, but still retains ability to increasetranscription of an operably linked polynucleotide, especially celltype-specific transcription activity. The difference in a TRE can be dueto differences in linear sequence, arising from, for example, single ormultiple base mutation(s), addition(s), deletion(s), and/ormodification(s) of the bases. The difference can also arise from changesin the sugar(s), and/or linkage(s) between the bases of a TRE.

[0067] Certain point mutations within sequences of TREs have been shownto decrease transcription factor binding and gene activation. One ofskill in the art would recognize that some alterations of bases in andaround known the transcription factor binding sites are more likely tonegatively affect gene activation and cell-specificity, whilealterations in bases which are not involved in transcription factorbinding are not as likely to have such effects. Certain mutations arealso capable of increasing TRE activity. Testing of the effects ofaltering bases may be performed in vitro or in vivo by any method knownin the art, such as mobility shift assays, or transfecting vectorscontaining these alterations in TRE functional and TRE non-functionalcells. Additionally, one of skill in the art would recognize that pointmutations and deletions can be made to a TRE sequence without alteringthe ability of the sequence to regulate transcription.

[0068] A “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of any polynucleotide(s) and/or vector(s)of this invention. Host cells include progeny of a single host cell, andthe progeny may not necessarily be completely identical (in morphologyor of total DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation and/or change. A host cell includescells transfected or infected in vivo with a polynucleotide and/or avector of this invention.

[0069] As used herein, the terms “neoplastic cells”, “neoplasia”,“tumor”, “tumor cells”, “cancer” and “cancer cells”, (usedinterchangeably) refer to cells which exhibit relatively autonomousgrowth, so that they exhibit an aberrant growth phenotype characterizedby a significant loss of control of cell proliferation. Neoplastic cellscan be malignant or benign.

[0070] In the context of a viral vector, e.g., adenovirus vector(s), ofthe invention, a “heterologous” promoter or enhancer is one which is notpresent in wild-type virus. Examples of a heterologous promoter orenhancer are the albumin promoter or enhancer and other viral promotersand enhancers, such as SV40.

[0071] In the context of adenovirus vector(s), an “endogenous” promoter,enhancer, or TRE is native to or derived from adenovirus.

[0072] The term “gene” is well understood in the art and is apolynucleotide encoding a polypeptide. In addition to the polypeptidecoding regions, a gene includes non-coding regions including, but notlimited to, introns, transcribed but untranslated segments, andregulatory elements upstream and downstream of the coding segments.

[0073] In the context of adenovirus vector(s), a “heterologouspolynucleotide” or “transgene” is any gene that is not present inwild-type adenovirus. Preferably, the transgene will also not beexpressed or present in the target cell prior to introduction by theadenovirus vector. Examples of preferred transgenes are provided below.

[0074] A sequence, whether polynucleotide or polypeptide, “depicted in”a SEQ ID NO, means that the sequence is present as an identicalcontiguous sequence in the sequence of the SEQ ID NO.

[0075] The terms “polynucleotide” and “nucleic acid”, usedinterchangeably herein, refer to a polymeric form of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides. These termsinclude a single-, double- or triple-stranded DNA, genomic DNA, cDNA,RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically, biochemically modified, non-naturalor derivatized nucleotide bases. The following are non-limiting examplesof polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA,rRNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs, uracyl, other sugars and linkinggroups such as fluororibose and thioate, and nucleotide branches. Thesequence of nucleotides may be interrupted by non-nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component. Other types of modificationsincluded in this definition are caps, substitution of one or more of thenaturally occurring nucleotides with an analog, and introduction ofmeans for attaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

[0076] A polynucleotide or polynucleotide region has a certainpercentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity”to another sequence means that, when aligned, that percentage of basesare the same in comparing the two sequences. This alignment and thepercent homology or sequence identity can be determined using softwareprograms known in the art, for example those described in CurrentProtocols in Molecular Biology (F. M. Ausubel et al., eds., 1987)Supplement 30, section 7.7.18, Table 7.7.1. A preferred alignmentprogram is ALIGN Plus (Scientific and Educational Software,Pennsylvania), preferably using default parameters.

[0077] “Androgen receptor”, or AR as used herein refers to a proteinwhose function is to specifically bind to androgen and, as a consequenceof the specific binding, recognize and bind to an androgen responseelement (ARE), following which the AR is capable of regulatingtranscriptional activity. The AR is a nuclear receptor that, whenactivated, binds to cellular androgen-responsive element(s). In normalcells the AR is activated by androgen, but in non-normal cells(including malignant cells) the AR may be activated by non-androgenicagents, including hormones other than androgens. Encompassed in the term“androgen receptor” are mutant forms of an androgen receptor, as long asthe function is sufficiently preserved. Mutants include androgenreceptors with amino acid additions, insertions, truncations anddeletions, as long as the function is sufficiently preserved. In thiscontext, a functional androgen receptor is one that binds both androgenand, upon androgen binding, an ARE.

[0078] “Replication” and “propagation” are used interchangeably andrefer to the ability of an adenovirus vector of the invention toreproduce or proliferate. This term is well understood in the art. Forpurposes of this invention, replication involves production ofadenovirus proteins and is generally directed to reproduction ofadenovirus. Replication can be measured using assays standard in the artand described herein, such as a burst assay or plaque assay.“Replication” and “propagation” include any activity directly orindirectly involved in the process of virus manufacture, including, butnot limited to, viral gene expression, production of viral proteins,nucleic acids or other components, packaging of viral components intocomplete viruses, and cell lysis.

[0079] A “gene essential for replication” is a gene whose transcriptionis required for the vector to replicate in a cell.

[0080] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Theseterms also include proteins that are post-translationally modifiedthrough reactions that include glycosylation, acetylation andphosphorylation.

[0081] As used herein, “cytotoxicity” is a term well understood in theart and refers to a state in which one or more of a cell's usualbiochemical or biological functions are perturbed (i.e., inhibited orelevated). These activities include, but are not limited to, metabolism,cellular replication, DNA replication, transcription, translation, anduptake of molecules. “Cytotoxicity” includes cell death and/orcytolysis. Assays are known in the art which indicate cytotoxicity, suchas dye exclusion, ³H-thymidine uptake, and plaque assays. The term“selective cytotoxicity”, as used herein, refers to the cytotoxicityconferred by an adenoviral vector of the present invention on a cellwhich allows a cell type-specific TRE to function when compared to thecytotoxicity conferred by an adenoviral vector of the present inventionon,a cell which does not allow, or is less permissive for, the same TREto function. Such cytotoxicity may be measured, for example, by plaqueassays, reduction or stabilization in size of a tumor comprising targetcells, or the reduction or stabilization of serum levels of a markercharacteristic of the tumor cells or a tissue-specific marker, e.g., acancer marker such as prostate specific antigen.

[0082] As used herein, a “cytotoxic” gene is a gene whose expression ina cell, either alone or in conjunction with adenovirus replication,enhances the degree and/or rate of cytotoxic and/or cytolytic activityin the cell.

[0083] A “therapeutic” gene is a gene whose expression in a cell isassociated with a desirable result. In the cancer context, thisdesirable result may be, for example, cytotoxicity, repression orslowing of cell growth, and/or cell death.

[0084] A “biological sample” encompasses a variety of sample typesobtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

[0085] An “individual” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, rodents,primates, farm animals, sport animals, and pets.

[0086] An “effective amount” is an amount sufficient to effectbeneficial or desired clinical results. An effective amount can beadministered in one or more administrations. For purposes of thisinvention, an effective amount of an adenoviral vector is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse, slow or delaythe progression of the disease state.

[0087] As used herein, “treatment” is an approach for obtainingbeneficial or desired clinical results. For~purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing spread (i.e.,metastasis) of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment.

[0088] “Palliating” a disease means that the extent and/or undesirableclinical manifestations of a disease state are lessened and/or timecourse of the progression is slowed or lengthened, as compared to notadministering adenoviral vectors of the present invention.

[0089] A “masked adenovirus” is an adenovirus which has been complexedwith a hydrophilic polymer (“masking agent”). The adenovirus may be anyadenovirus, including naturally occurring isolates of adenovirus orengineered adenovirus vectors such as those disclosed in the instantapplication. Masking agents are preferably of low immunogenicity.Examples of acceptable hydrophilic polymers includepolyethylene/polypropylene copolymers, polyacrylic acid analogues, sugarpolymers such as cellulose, polyformaldehyde, poly (N-vinylpyrolidone),polyethylene glycol (PEG), and the like. The hydrophilic polymer may becomplexed by covalent or non-covalent attachment to the capsid proteinsof the virus, particularly the hexon and fiber capsid proteins. Apreferred hydrophilic polymer is PEG, and a preferred masked adenovirusis PEG covalently linked to adenovirus (“covalently pegylatedadenovirus”).

[0090] Adenoviral Vectors of the Invention

[0091] Replication-competent adenovirus vehicles are provided. Theviruses comprise at least one gene under the transcriptional control ofa transcriptional initiation region (transcriptional regulatory, orresponse element, TRE) specifically regulated by target host cells. Thegenes that are regulated by the specifically regulated transcriptionalinitiation region may be early or late adenovirus genes and/ortransgenes. By providing for regulated transcription restricted tospecific host cell targets, one can provide for adenoviruses that can beused as vehicles for introducing genetic capability into host targetcells, as distinct from other host cell types. The transgenes serve tomodify the genotype or phenotype of the target cell, in addition to anymodification of the genotype or phenotype resulting from the presence ofthe adenovirus. With competent adenoviruses, proliferation of theadenovirus may be used for its cytotoxic effect.

[0092] There are a number of different types of adenovirus, such as Ad2,Ad5, and Ad40, which may differ to minor or significant degrees.Particularly, Ad5 and Ad40 differ as to their host cell tropism, as wellas the nature of the disease induced by the virus. For the purpose ofthe subject invention, Ad5 will be exemplified.

[0093] The genes of the adenovirus that are of interest for the subjectinvention may be divided into two groups, the early genes and the lategenes, the expression of the latter being controlled by the major latepromoter. Of the early genes, there are E1A, E1B, E2, E3 and E4. The E1Agene is expressed immediately after viral infection (0-2 h) and beforeany other viral genes. E1A protein acts as a trans-actingpositive-acting transcriptional regulatory factor, and is required forthe expression of the other early viral genes and the promoter proximalmajor late genes. Despite the nomenclature, the promoter proximal genesdriven by the major late promoter are expressed during early times afterAd5 infection. In the absence of a functional E1A gene, viral infectiondoes not proceed, because the gene products necessary for viral DNAreplication are not produced.

[0094] The E1B protein functions in trans and is necessary for transportof late mRNA from the nucleus to the cytoplasm. Defects in E1Bexpression result in poor expression of late viral proteins and aninability to shut off host cell protein synthesis.

[0095] The E4 gene has a number of transcription products. Open readingframes (ORF) 3 and ORF 6 of the E4 transcription unit increase theaccumulation of major late transcription unit mRNAs by binding the55-kDa protein from E1B and heterodimers of E2F-1 and DP-1. In theabsence of functional protein from ORF3 and ORF6, plaques are producedwith an efficiency less than 10⁻⁶ of that of wild type virus.

[0096] The major late genes relevant to the subject invention are genessuch as L1, L2 and L3, which encode proteins of the AD5 virus virion.

[0097] Regions of the adenovirus which may be deleted, usually at least500 nt, more usually at least about 1 knt, include in the AD5 genomenucleotides 300 to 3600 in E1, particularly 342 to 3523; 27000 to 31000,particularly 28133 to 30818 or 27865 to 30995 in E3. The deletion willbe at least sufficient for insertion of the desired construct and allowfor packaging.

[0098] The subject vectors can be used for a wide variety of purposes.The purpose will vary with the target cell. Suitable target cells arecharacterized by the transcriptional activation of the cell specifictranscriptional response element in the adenovirus vehicle. Thetranscription initiation region will usually be activated in less thanabout 5%, more usually less than about 1%, and desirably by less thanabout 0.1% of the cells in the host.

[0099] Regulation of transcriptional activation is the result ofinteraction between transcriptional activators bound to cis-regulatoryelements, factors bound to basal transcriptional elements and theactivity of transcriptional mediators, or coactivators. The absence orpresence of any of these factors may affect the level of transcription.Additionally, factors may be present in an inactive form, where thefactors are activated through chemical modification, particularly as theresult of a cellular signaling mechanism. In some cases, signalingmolecules are able to act directly to activate transcription. Any ofthese mechanisms may operate to limit the types of cells in which thevehicle transcription initiation region is active.

[0100] It will be understood by one of skill in the art that very lowbasal levels of transcription may be present in non-targeted cell types.By transcriptional activation, it is intended that transcription will beincreased above basal levels in the target cell by at least about 100fold, more usually by at least about 1000 fold.

[0101] The cell specific response element, also referred to herein as acell type-specific transcriptional response element (TRE), may be usedwith an adenovirus gene that is essential for propagation, so thatreplication competence is only achievable in the target cell, and/orwith a transgene for changing the phenotype of the target cell. Bytransgene it is intended any gene that is not present in wild-typeadenovirus, frequently the transgene will also not be expressed in thetarget cell, prior to introduction by the adenovirus.

[0102] As exemplified by employing a cell specific response elementcomprising a promoter and enhancer construct specific for prostatecells, various genetic capabilities may be introduced into prostatecells expressing prostate specific antigen. Of particular interest isthe opportunity to introduce cytotoxic effects that are controlled by atranscriptional initiation region specifically active in prostate cells.Other cell types that have specific active transcription factorsassociated with a state for which modulation is desirable includeleukocytes, particularly lymphocytes, epithelial cells, endothelialcells, hepatic cells, pancreatic cells, neuronal cells, andkeratinocytes. Since the adenovirus results in transient expression(approximately 6 to 8 weeks), one can provide transient capability tocells, where the desired result only requires a limited period forresponse.

[0103] Accordingly, the invention provides an adenovirus vectorcomprising an adenovirus gene under transcriptional control of a celltype-specific TRE. In some embodiments, a first cell type-specifictranscriptional response element controls expression of a firstadenovirus gene, and a second cell type-specific transcriptionalresponse element controls expression of a second gene. The genes to becontrolled under these TREs are preferably adenoviral genes essentialfor propagation, more preferably early genes. Alternatively, the genesto be controlled under these TREs may be a gene essential forpropagation and a transgene.

[0104] Accordingly, the invention includes adenovirus vectors comprisinga first adenovirus gene under transcriptional control of a firsttranscriptional regulatory element (TRE) and at least a second geneunder transcriptional control of a second TRE, wherein the first TRE andsecond TRE are cell (i.e., cell or tissue) specific. It is understoodthat there may or may not be additional TREs in these adenoviralvectors, and that these additional TREs may or may not be substantiallyidentical to the first and/or second TREs. Accordingly, the inventionincludes use of three or more, four or more, TREs.

[0105] A cell type-specific TRE can also comprise multimers. Forexample, a cell type-specific TRE can comprise a tandem series of atleast two, at least three, at least four, or at least five TREs. Thesemultimers may also contain heterologous promoter and/or enhancersequences.

[0106] A cell type-specific TRE may or may not lack a silencer. Thepresence of a silencer (i.e., a negative regulatory element) may assistin shutting off transcription (and thus replication) in non-permissivecells (i.e., cells in a normal cell state). Thus, the presence of asilencer may confer enhanced cell status-specific replication by moreeffectively preventing adenoviral vector replication in non-targetcells. Alternatively, the lack of a silencer may assist in effectingreplication in target cells, thus conferring enhanced cell type-specificreplication due to more effective replication in target cells.

[0107] In one embodiment, two substantially identical TREs controltranscription of adenovirus early genes, preferably E1A and E1B. It isunderstood, however, that any of a number of combinations of genes maybe used with these at least two TREs. Other preferred embodimentsinclude those which contain substantially identical TREs that driveexpression of E1A, E1B, and E4. Such constructs may or may notadditionally contain a transgene, which may or may not be under controlof a substantially identical TRE. Preparation of these and otherembodiments are provided below and in the examples.

[0108] In embodiments in which two substantially identical celltype-specific TREs are used, the invention does not require that theTREs be derived from the same gene. As long as the TRE sequences aresubstantially identical, and the requisite functionality is displayed,the TREs may be derived from different genes.

[0109] In another embodiment, the adenovirus vectors of the inventioncomprise a first adenovirus gene under the transcriptional control of acell-specific heterologous TRE and at least one other gene, such as anadenoviral gene or a transgene, under control of another heterologousTRE which is different from the first TRE, where the heterologous TREsare functional in the same cell but are not the same in polynucleotidesequence (i.e., have different polynucleotide sequences). Preferably, atleast two of the heterologous TREs in an adenovirus vector are cellspecific for the same cell. Preferably, the adenovirus gene is one thatenhances cell death, more preferably one that is essential foradenovirus replication. Preferably, at least one of the adenovirus genesnecessary for cell replication is an early gene and the genes undertranscriptional control of the heterologous TREs are necessary forreplication. By providing for cell-specific transcription through theuse of multiple heterologous TREs, the invention provides adenovirusvectors that can be used for cell-specific cytotoxic effects due toselective replication. It has been demonstrated that adenovirus vectorswhich include at least two different heterologous TREs are more stableand provide greater cell specificity with regard to replication thanpreviously described adenovirus vectors. Accordingly, adenovirus vectorshave been constructed in which each of the E1A and E1B genes are undertranscriptional control of two different heterologous. It is understood,however, that any of a number of combinations of genes may be used withany combination of at least two TREs (as further described below).Further preferred embodiments include those which contain at least twodifferent heterologous TREs that drive expression of E1A, E1B, and E4.Such constructs may or may not additionally contain a transgene, whichmay or may not be under control of a TRE. See, e.g., InternationalPatent Application Nos. WO 98/39466 and WO 98/39464.

[0110] Cell Type-Specific Transcriptional Response Elements

[0111] Depending upon the target cell type, various enhancers may beused to provide for target cell specific transcription. Withlymphocytes, for B cells one may use the Ig enhancer, for T cells onemay use the T cell antigen receptor promoter. For the different musclecells, one may use the promoters for the different myosins. Forendothelial cells, one may use the different promoters for the differentselectins. For each type of cell, there will be specific proteinsassociated with the cell, which allows for target cell specifictranscription.

[0112] In one embodiment, the invention includes adenovirus vectorswherein the substantially identical TREs are prostate cell specific. Forexample, TREs that function preferentially in prostate cells and can beused in the present invention to target adenovirus replication toprostate neoplasia, include, but are not limited to, TREs derived fromthe prostate-specific antigen gene (PSA-TRE), the glandular kallikrein-1gene (from the human gene, hKLK2-TRE), and the probasin gene (PB-TRE).All three of these genes are preferentially expressed in prostate cellsand the expression is androgen-inducible. Generally, expression of genesresponsive to androgen induction requires the presence of an androgenreceptor (AR).

[0113] The region of the PSA gene that is used to provide cellspecificity dependent upon androgens, particular in prostate cells,involves approximately 6.0 kilobases. Schuur et al. (1996) J. Biol.Chem. 271:7043-7051. An enhancer region of approximately 1.5 kb inhumans is located between nt −5322 and nt −3739, relative to thetranscription start site of the PSA gene. The PSA promoter consists ofthe sequence from about nt −540 to nt +8 relative to the transcriptionstart site. Juxtapositioning of these two genetic elements yields afully functional, minimal prostate-specific enhancer/promoter (PSE) TRE.Other portions of the approximately 6.0 kb region of the PSA gene can beused in the present invention, as long as requisite functionality ismaintained.

[0114] The PSE and PSA TRE depicted in (SEQ ID NO: 1) is the same asthat given in GenBank Accession No. U37672, and published. Schuur et al.(1996). A variant PSA-TRE nucleotide sequence is depicted in (SEQ ID NO:2). This is the PSA-TRE contained within CN706 clone 35.190.13. CN706 isan adenoviral vector in which the E1A gene in Ad5 is undertranscriptional control of a PSA-TRE. CN706 demonstrates selectivecytotoxicity toward PSA-expressing cells in vitro and in vivo. Rodriguezet al. (1997). CN706 was passaged through 293 and LNCaP cells. A clone,designated 35.190.13 was isolated. The structure of this clone wasconfirmed by PCR, restriction endonuclease digestion and Southernblotting. Both DNA strands of the CN706 clone 35.190.13 were sequencedbetween positions 1 and 3537. Seven single base pair changes were foundin the PSE, compared to the sequence reported by Schuur et al. (1996).These point mutations are not in the ARE and are thus not likely toaffect the function of the enhancer. One mutation was found in the PSApromoter region, but is not likely to affect gene expression from thispromoter. In addition to these mutations, a missense mutation was foundin the first exon of E1A. This C to G transition at position 3032results in a Glu to Arg change in the E1A protein sequence. Thismutation does not appear to diminish E1A function.

[0115] The region that is employed to provide cell specificity dependentupon androgens, particularly in prostate cells, involves anapproximately 1.5 kb enhancer region and a 0.5 kb promoter region. Theenhancer region in humans is located between nt −5322 and nt −3739,relative to the transcription start site of the prostate specificantigen (PSA) gene. The promoter consists of nt −540 to nt +8.Juxtaposition of the two genetic elements yields a fully functional,minimal prostate-specific enhancer promoter (PSE). The enhancer containsthree regions that bind prostate-specific DNA binding proteins, one ofwhich contains a putative androgen response element. The promoter regioncontains typical TATA and CAAT boxes as well as a second putativeandrogen response element.

[0116] Human glandular kallikrein (hKLK2, encoding the hK2 protein) isexpressed exclusively in the prostate and its expression is up-regulatedby androgens primarily by transcriptional activation. Wolf et al. (1992)Molec. Endocrinol. 6:753-762. Morris (1989) Clin. Exp. Pharm. Physiol.16:345-351; Qui et al. (1990) J. Urol. 144:1550-1556; Young et al.(1992) Biochem. 31:818-824. The levels of hK2 found in various tumorsand in the serum of patients with prostate cancer differ substantiallyfrom those of PSA and indicate that hK2 antigen may be a significantmarker for prostate cancer. Circulating hK2 in different relativeproportions to PSA has been detected in the serum of patients withprostate cancer. Charlesworth et al. (1997) Urology 49:487-493.Expression of hK2 has been detected in each of 257 radical prostatectomyspecimens analyzed. Darson et al. (1997) Urology 49:857-862. Theintensity and extent of hK2 expression, detected using specificantibodies, increased from benign epithelium to high-grade prostaticintraepithelial neoplasia (PIN) and adenocarcinoma, whereas PSA andprostate acid phosphatase displayed an inverse pattern ofimmunoreactivity. Darson et al. (1997). Indeed, it has been reportedthat a certain percentage of PSA-negative tumors have detectable hK2.Tremblay et al. (1997) Am. J. Pathol. 150:455-459.

[0117] The activity of the hKLK2 5′ promoter has been previouslydescribed and a region up to −2256 relative to the transcription startsite was previously disclosed. Schedlich et al. (1987) DNA 6:429-437.The hKLK2 promoter is androgen responsive and, in plasmid constructswherein the promoter alone controls the expression of a reporter gene,expression of the reporter gene is increased approximately 10-fold inthe presence of androgen. Murtha et al. (1993) Biochem. 32:6459-6464.hKLK2 enhancer activity is found within a polynucleotide sequenceapproximately nt −12,014 to nt −2257 relative to the start oftranscription (depicted in SEQ ID NO: 3) and, when this sequence isoperably linked to an hKLK2 promoter and a reporter gene, transcriptionof operably-linked sequences in prostate cells increases in the presenceof androgen at levels approximately 30- to approximately 100-fold overthe level of transcription in the absence of androgen. This induction isgenerally orientation independent and position independent. Enhanceractivity has also been demonstrated in the following regions (allrelative to the transcription start site): about nt −3993 to about nt−3643 (nt 8021 to 8371 of SEQ ID NO: 3), about nt −4814 to about nt−3643 (nt 7200 to 8371 of SEQ ID NO: 3), about nt −5155 to about nt−3387 (nt 6859 to 8627 of SEQ ID NO: 3), about nt −6038 to about nt−2394 (nt 5976 to 9620 of SEQ ID NO: 3).

[0118] Thus, an hKLK2 enhancer can be operably linked to an hKLK2promoter or a heterologous promoter to form an hKLK2 transcriptionalregulatory element (hKLK2-TRE). An hKLK2-TRE can then be operably linkedto a heterologous polynucleotide to confer hKLK2-TRE-specifictranscriptional regulation on the linked gene, thus increasing itsexpression.

[0119] The rat probasin (PB) gene encodes a nuclear and secretedprotein, probasin, that is only expressed in the dorsolateral prostate.Dodd et al. (1983) J. Biol. Chem. 258:10731-10737; Matusik et al. (1986)Biochem. Cell. Biol. 64: 601-607; and Sweetland et al. (1988) Mol. Cell.Biochem. 84: 3-15. The dorsolateral lobes of the murine prostate areconsidered the most homologous to the peripheral zone of the humanprostate, where approximately 68% of human prostate cancers are thoughtto originate.

[0120] A PB-TRE has been shown in an approximately 0.5 kb fragment ofsequence upstream of the probasin coding sequence, from about nt −426 toabout nt +28 relative to the transcription start site, as depicted in(SEQ ID NO: 4). This minimal promoter sequence from the PB gene appearsto provide sufficient information to direct development andhormone-regulated expression of an operably linked heterologous genespecifically to the prostate in transgenic mice. Greenberg et al. (1994)Mol. Endocrinol. 8:230-239.

[0121] In the present invention, replication-competent adenovirusvectors directed at specific target cells may also be generated with theuse of TREs that are preferentially functional in the target tumorcells. Non-limiting examples of tumor cell-specific TREs, andnon-limiting examples of respective potential target cells, include TREsfrom the following genes: α-fetoprotein (AFP) (liver cancer); mucin-likeglycoprotein DF3 (MUC1) (breast carcinoma); carcinoembryonic antigen(CEA) (colorectal, gastric, pancreatic, breast, and lung cancers);plasminogen activator urokinase (uPA) and its receptor gene (breast,colon, and liver cancers); and HER-2/neu (c-erbB2/neu) (breast, ovarian,stomach, and lung cancers).

[0122] AFP is an oncofetal protein, the expression of which is primarilyrestricted to developing tissues of endodermal origin (yolk sac, fetalliver, and gut), although the level of its expression varies greatlydepending on the tissue and the developmental stage. AFP is of clinicalinterest because the serum concentration of AFP is elevated in amajority of hepatoma patients, with high levels of AFP found in patientswith advanced disease. The serum AFP levels in patients appear to beregulated by AFP expression in hepatocellular carcinoma but not insurrounding normal liver. Thus, the AFP gene appears to be regulated tohepatoma cell-specific expression.

[0123] Cell-specific TREs from the AFP gene have been identified. Forexample, the cloning and characterization of human AFP-specific enhanceractivity is described in Watanabe et al. (1987) J. Biol. Chem.262:4812-4818. The entire 5′ AFP flanking region (containing thepromoter, putative silencer, and enhancer elements) is contained withinapproximately 5 kb upstream from the transcription start site (SEQ IDNO: 5).

[0124] The AFP enhancer region in human is located between about nt−3954 and about nt −3335, relative to the transcription start site ofthe AFP gene. The human AFP promoter encompasses a region from about nt−174 to about nt +29. Juxtapositioning of these two genetic elements, asdepicted in SEQ ID NO: 6, yields a fully functional AFP-TRE. Ido et al.(1995) describe a 259 bp promoter fragment (nt −230 to nt +29) that is,specific for HCC. Cancer Res. 55:3105-3109. The AFP enhancer containstwo regions, denoted A and B, located between nt −3954 and nt −3335relative to the transcription start site. The promoter region containstypical TATA and CAAT boxes. Preferably, the AFP-TRE contains at leastone enhancer region. More preferably, the AFP-TRE contains both enhancerregions.

[0125] Suitable target cells for adenoviral vectors containing AFP-TREsare any cell type that allow an AFP-TRE to function. Preferred are cellsthat express, or produce, AFP, including, but not limited to, tumorcells expressing AFP. Examples of such cells are hepatocellularcarcinoma cells, gonadal and other germ cell tumors (especiallyendodermal sinus tumors), brain tumor cells, ovarian tumor cells, acinarcell carcinoma of the pancreas (Kawamoto et al. (1992)Hepatogastroenterology 39:282-286), primary gall bladder tumor(Katsuragi et al. (1989) Rinsko Hoshasen 34:371-374), uterineendometrial adenocarcinoma cells (Koyama et al. (1996) Jpn. J CancerRes. 87:612-617), and any metastases of the foregoing (which can occurin lung, adrenal gland, bone marrow, and/or spleen). In some cases,metastatic disease to the liver from certain pancreatic and stomachcancers produce AFP. Especially preferred are hepatocellular carcinomacells and any of their metastases. AFP production can be measured usingassays standard in the art, such as RIA, ELISA or Western blots(immunoassays) to determine levels of AFP protein production or Northernblots to determine levels of AFP mRNA production. Alternatively, suchcells can be identified and/or characterized by their ability toactivate transcriptionally an AFP-TRE (i.e., allow an AFP-TRE tofunction).

[0126] The protein urokinase plasminogen activator (uPA) and its cellsurface receptor, urokinase plasminogen activator receptor (uPAR), areexpressed in many of the most frequently occurring neoplasia and appearto represent important proteins in cancer metastasis. Both proteins areimplicated in breast, colon, prostate, liver, renal, lung and ovariancancer. Transcriptional regulatory elements that regulate uPA and uPARtranscription have been extensively studied. Riccio et al. (1985)Nucleic Acids Res. 13:2759-2771; Cannio et al., (1991) Nucleic AcidsRes. 19:2303-2308.

[0127] CEA is a 180,000-Dalton glycoprotein tumor-associated antigenpresent on endodermally-derived neoplasia of the gastrointestinal tract,such as colorectal, gastric (stomach) and pancreatic cancer, as well asother adenocarcinomas such as breast and lung cancers. CEA is ofclinical interest because circulating CEA can be detected in the greatmajority of patients with CEA-positive tumors. In lung cancer, about 50%of total cases have circulating CEA, with high concentrations of CEA(greater than 20 ng/ml) often detected in adenocarcinomas. Approximately50% of patients with gastric carcinoma are serologically positive forCEA.

[0128] The 5′ upstream flanking sequence of the CEA gene has been shownto confer cell-specific activity. The CEA promoter region, approximatelythe first 424 nucleotides upstream of the translational start site inthe 5′ flanking region of the gene, was shown to confer cell-specificactivity when the region provided higher promoter activity inCEA-producing cells than in non-producing HeLa cells.. Schrewe et al.(1990) Mol. Cell. Biol. 10:2738-2748. In addition, cell-specificenhancer regions have been found. WO/95/14100. The entire 5′ CEAflanking region (containing the promoter, putative silencer, andenhancer elements) appears to be contained within approximately 14.5 kbupstream from the transcription start site. Richards et al. (1995); WO95/14100. Further characterization of the 5′ flanking region of the CEAgene by Richards et al. (1995) indicated two upstream regions, −13.6 to−10.7 kb or −6.1 to −4.0 kb, when linked to the multimerized promoterresulted in high-level and selective expression of a reporter constructin CEA-producing LoVo and SW1463 cells. Richards et al. (1995) alsolocalized the promoter region to nt −90 and nt +69 relative to thetranscriptional start site, with region nt −41 to nt −18 as essentialfor expression. WO95/14100 describes a series of 5′ flanking CEAfragments which confer cell-specific activity, such as about nt −299 toabout nt +69; about nt −90 to about nt +69; nt −14,500 to nt −10,600; nt−13,600 to nt −10,600, nt −6100 to nt −3800. In addition, cell specifictranscription activity is conferred on an operably linked gene by theCEA fragment from nt −402 to nt +69, depicted in (SEQ ID NO: 7). AnyCEA-TREs used in the present invention are derived from mammalian cells,including but not limited to, human cells. Thus, any of the CEA-TREs maybe used in the invention as long as requisite desired functionality isdisplayed in the adenovirus vector. The cloning and characterization ofCEA sequences have been described in the literature and are thus madeavailable for practice of this invention and need not be described indetail herein.

[0129] The protein product of the MUC1 gene (known as mucin or MUC1protein; episialin; polymorphic epithelial mucin or PEM; EMA; DF3antigen; NPGP; PAS-O; or CA15.3 antigen) is normally expressed mainly atthe apical surface of epithelial cells lining the glands or ducts of thestomach, pancreas, lungs, trachea, kidney, uterus, salivary glands, andmammary glands. Zotter et al. (1988) Cancer Rev. 11-12: 55-101; andGirling et al. (1989) Int. J. Cancer 43: 1072-1076. However, mucin isoverexpressed in 75-90% of human breast carcinomas. Kufe et al. (1984)Hybridoma 3: 223-232. For reviews, see Hilkens (1988) Cancer Rev. 11-12:25-54; and Taylor-Papadimitriou, et al. (1990) J. Nucl. Med. Allied Sci.34: 144-150. Mucin protein expression correlates with the degree ofbreast tumor differentiation. Lundy et al. (1985) Breast Cancer Res.Treat. 5: 269-276. This overexpression appears to be controlled at thetranscriptional level.

[0130] Overexpression of the MUC1 gene in human breast carcinoma cellsMCF-7 and ZR-75-1 appears to be regulated at the transcriptional level.Kufe et al. (1984); Kovarik (1993) J. Biol. Chem. 268:9917-9926; and Abeet al. (1990) J. Cell. Physiol. 143: 226-231. The regulatory sequencesof the MUC1 gene have been cloned, including the approximately 0.9 kbupstream of the transcription start site which contains a TRE thatappears to be involved in cell-specific transcription, depicted in SEQID NO: 8. Abe et al. (1993) Proc. Natl. Acad. Sci. USA 90: 282-286;Kovarik et al. (1993); and Kovarik et al. (1996) J. Biol. Chem.271:18140-18147.

[0131] Any MUC1-TREs used in the present invention are derived frommammalian cells, including but not limited to, human cells. Preferably,the MUC1-TRE is human. In one embodiment, the MUC1-TRE may contain theentire 0.9 kb 5′ flanking sequence of the MUC1 gene. In otherembodiments, the MUC1-TREs comprise the following sequences (relative tothe transcription start site of the MUC1 gene): about nt −725 to aboutnt +31, nt −743 to about nt +33, nt −750 to about nt +33, and nt −598 toabout nt +485 (operably-linked to a promoter).

[0132] The c-erbB2/neu gene (HER-2/neu or HER) is a transforming genethat encodes a 185 kD epidermal growth factor receptor-relatedtransmembrane glycoprotein. In humans, the c-erbB2/neu protein isexpressed during fetal development, however, in adults, the protein isweakly detectable (by immunohistochemistry) in the epithelium of manynormal tissues. Amplification and/or over-expression of the c-erbB2/neugene has been associated with many human cancers, including breast,ovarian, uterine, prostate, stomach and lung cancers. The clinicalconsequences of the c-erbB2/neu protein over-expression have been beststudied in breast and ovarian cancer. c-erbB2/neu proteinover-expression occurs in 20 to 40% of intraductal carcinomas of thebreast and 30% of ovarian cancers, and is associated with a poorprognosis in subcategories of both diseases. Human, rat and mousec-erbB2/neu TREs have been identified and shown to confer c-erbB2/neuexpressing cell specific activity. Tal et al. (1987) Mol. Cell. Biol.7:2597-2601; Hudson et al. (1990) J. Biol. Chem. 265:4389-4393;Grooteclaes et al. (1994) Cancer Res. 54:4193-4199; Ishii et al. (1987)Proc. Nati. Acad. Sci. USA 84:4374-4378; Scott et al. (1994) J. Biol.Chem. 269:19848-19858.

[0133] In the present invention, cell type-specific TREs which aretumor-specific may be used in conjunction with other, non-tumor-specificcell type-specific TREs from the following exemplary genes (tissue inwhich the TREs are specifically functional are in parentheses): vascularendothelial growth factor receptor,(endothelium); albumin (liver);factor VII (liver); fatty acid synthase (liver); von Willebrand factorand fit-1 (endothelium); alpha-actin and myosin heavy chain (both insmooth muscle); synthetase I (small intestine); Na—K—Cl transporter(kidney). Additional cell type-specific TREs are known in the art.

[0134] Additional tumor- and/or cell type-specific TREs known in the artinclude the following: aromatase, mammary gland-specific promoter,mammaglobin, urokinase, and human alpha-lactalbumin (breast tissue);BCSG1, BRCA1, and BRCA2 (breast cancer); human papilloma virus (HPV)cell type dependent regulatory element (cervical cancer); BLCA4 (bladdercancer); uroplakin (bladder); NCA (gastric cancer); hypoxanthinephosphoribosyltransferase (HPRT) (glioma); AVP, human pulmonarysurfactant protein B gene, and puromycin N-acetyltransferase (lungcancer); tyrosinase, gp100, tyrosinase related proteins 1 and 2; MART-1,and melanocyte specific factory (MSF) (melanoma); HER2/neu, urokinase,and CA125 (ovarian cancer); SL3-3 and T cell antigen receptor (T celllymphoma); and prostatic acid phosphatase (prostate). Descriptions ofthese cell-specific TREs can be found in various publications, includingthe following: Zhou et al. (1996) J. Biol. Chem. 271:15164-15202(aromatase); International Patent Application No. WO 98/15634 (mammarygland-specific promoter); Watson et al. (1996) Cancer Res. 56:860-865(mammaglobin); Ji et al. (1997) Cancer Res. 57:759-764 (breastcancer-specific gene BCSG1); (1995) Gene 159:65-71 (HER-2/neu); Cannioet al. (1991) Nucl. Acids Res. 19:2303-2308 (urokinase); (1993) Virol.195:500-510 (HPV cell type dependent regulatory element); Rincon-Limaset al. (1994) J. Neurosci. Res. 38:259-267 (HPRT); (1992) Gene117:255-258 (puromycin N-acetyltransferase); Bohinski et al. (1993) J.Biol. Chem. 268:11160-11166 (human pulmonary surfactant protein B gene);Vile et al. (1993) Cancer Res. 53:3860-3864 (tyrosinase); Butterfield etal. (1997) Gene 191:129-134 (MART-1); Yavuzer et al. (1994) Mol. Cell.Biol. 14:3494-3503 (MSF); Garcia-Arenas et al. (1995) Mol. Cell.Endocrinol. 111:29-37 (prostatic acid phosphatase); Boral et al. (1989)J. Virol. 63:76-84 (SL3-3); and (1990) Science 247:1225-1229.

[0135] The TREs listed above are provided as non-limiting examples ofTREs that would function in the instant invention. Additionalcell-specific TREs are known in the art, as are methods to identify andtest cell specificity of suspected TREs.

[0136] Activity of a TRE can be determined as follows. A TREpolynucleotide sequence or set of such sequences can be generated usingmethods known in the art, such as chemical synthesis, site-directedmutagenesis, PCR, and/or recombinant methods. The sequence(s) to betested can be inserted into a vector containing a promoter (if nopromoter element is present in the TRE) and an appropriate reporter geneencoding a reporter protein, including, but not limited to,chloramphenicol acetyl transferase (CAT),

-galactosidase, (encoded by the lacZ gene), luciferase (encoded by theluc gene), alkaline phosphatase, green fluorescent protein, and horseradish peroxidase. Such vectors and assays are readily available, from;inter alia, commercial sources. Plasmids thus constructed aretransfected into a suitable host cell to test for expression of thereporter gene as controlled by the putative TRE using transfectionmethods known in the art, such as calcium phosphate precipitation,electroporation, liposomes (lipofection), and DEAE dextran.

[0137] After introduction of the TRE-reporter gene construct into a hostcell under appropriate conditions, TRE activity may be measured bydetection and/or quantitation of reporter gene-derived mRNA or proteinproduct. The reporter gene protein can be detected directly (e.g.,immunochemically) or through its enzymatic activity, if any, with anappropriate substrate. Generally, to determine cell specific activity ofa TRE, the TRE-reporter gene constructs are introduced into a variety ofcell types. The amount of TRE activity is determined in each cell typeand compared to that of a reporter gene construct without the TRE. A TREis cell specific when it is preferentially functional in a specific typeof cell over a different type of cell.

[0138] For example, the specificity of PB-TRE activity for prostate cellthat express the androgen receptor (AR) was demonstrated as follows. Theregion of the PB 5′-flanking DNA (nt −426 to nt +28) (SEQ ID NO: 9)including the endogenous promoter sequences was inserted upstream of thefirefly luciferase gene to generate a chimeric PB-TRE-luc plasmid.Cationic-mediated, transient transfection of LNCaP (PSA-producing andAR-producing prostate carcinoma cells) and PC-3 (PSA-deficient andAR-deficient prostate carcinoma cells) cells was performed. The resultsshowed that LNCaP cells transfected with PB-TRE-luc had approximately400 times more activity than untransfected cells, indicating that thePB-TRE was intact. Further, the overall luciferase activity recovered inthe cellular extracts of transfected LNCaP cells was about 30-40-foldhigher than that measured in the cellular extracts of transfected PC-3cells. Thus, the results indicate that PB-TRE expression ispreferentially functional in PSA-producing, AR-producing prostatecarcinoma cells as compared to PSA-deficient, AR-deficient prostatecarcinoma cells and that PB-TRE is capable of mediating specificexpression in cells producing the androgen receptor.

[0139] Transgenes

[0140] Use of competent adenovirus, which is competent in particulartarget cells, allow for proliferation of the adenovirus in the targetcells resulting in the death of the host cells and proliferation of theadenovirus to other host cells. To further ensure cytotoxicity, one mayhave one or more transgenes present which have cytotoxic effect. In thisway one can provide high confidence that the target cells will bedestroyed while providing for the appropriate level of expression of thecytotoxic agents).

[0141] Accordingly, the adenovirus vectors of this invention can furtherinclude a heterologous polynucleotide (transgene) under the control of acell type-specific TRE. In this way, various genetic capabilities may beintroduced into target cells. For example, in certain instances, it maybe desirable to enhance the degree and/or rate of cytotoxic activity,due to, for example, the relatively refractory nature or particularaggressiveness of the target cell. This could be accomplished bycoupling the cell-specific replicative cytotoxic activity withcell-specific expression of, one or more metabolic enzymes such asHSV-tk, nitroreductase, cytochrome P450 or cytosine deaminase (cd) whichrender cells capable of metabolizing 5-fluorocytosine (5-FC) to thechemotherapeutic agent 5-fluorouracil (5-FU). This type of transgenesmay also be used to confer a bystander effect.

[0142] Transgenes that may be introduced into an adenovirus vector ofthe invention also include a factor capable of initiating apoptosis,antisense or ribozymes, which among other capabilities may be directedto mRNAs encoding proteins essential for proliferation, such asstructural proteins, transcription factors, polymerases, etc., viral orother pathogenic proteins, where the pathogen proliferatesintracellularly, cytotoxic proteins, e.g., the chains of diphtheria,ricin, abrin, etc., genes that encode an engineered cytoplasmic variantof a nuclease (e.g., RNase A) or protease (e.g., trypsin, papain,proteinase K, carboxypeptidase, etc.), or encode the Fas gene, and thelike. Other genes of interest include cytokines, antigens, transmembraneproteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF,IFN-α, -β, -γ, TNF-α, -β, TGF-α, -β, NGF, and the like. Further examplesinclude, proapoptotic genes such as Fas, Bax, Caspase, Fas ligands, andthe like; fusion genes which can lead to cell fusion or facilitate cellfusion such as V22, VSV and the like; tumor suppressor gene such as p53,RB, p16, p17, W9 and the like; genes associated with the cell cycle andgenes which encode anti-angiogenic proteins such as endostatin,angiostatin, and the like.

[0143] Other opportunities for specific genetic modification include Tcells, such as tumor infiltrating lymphocytes (TILs), where the TILs maybe modified to enhance expansion, enhance cytotoxicity, reduce responseto proliferation inhibitors, enhance expression of lymphokines, etc. Onemay also wish to enhance target cell vulnerability by providing forexpression of specific surface membrane proteins, e.g., B7, SV40 Tantigen mutants, etc.

[0144] In some embodiments, the adenovirus death protein (ADP), encodedwithin the E3 region, is maintained (i.e., contained) in the adenovirusvector. The ADP gene, under control of the major late promoter (MLP),appears to code for a protein (ADP) that is important in expediting hostcell lysis. Tollefson et al. (1996) J. Virol. 70(4):2296; Tollefson etal. (1992) J. Virol. 66(6):3633. Thus, adenoviral vectors containing theADP gene may render the adenoviral vector more potent, making possiblemore effective treatment and/or a lower dosage requirement.

[0145] Accordingly, the invention provides adenovirus vectors in whichan adenovirus gene is under transcriptional control of a first celltype-specific TRE and a polynucleotide sequence encoding an ADP undercontrol of a second cell type-specific TRE, and wherein preferably theadenovirus gene is essential for replication. A DNA sequence encoding anADP and the amino acid sequence of an ADP are depicted in SEQ ID NO: 10and SEQ ID NO: 11, respectively. Briefly, an ADP coding sequence isobtained preferably from Ad2 (since this is the strain in which ADP hasbeen more fully characterized) using techniques known in the art, suchas PCR. Preferably, the Y leader (which is an important sequence forcorrect expression of late genes) is also obtained and ligated to theADP coding sequence. The ADP coding sequence (with or without the Yleader) can then be introduced into the adenoviral genome, for example,in the E3 region (where the ADP coding sequence will be driven by theMLP). The ADP coding sequence could also be inserted in other locationsof the adenovirus genome, such as the E4 region. Alternatively, the ADPcoding sequence could be operably linked to a different type of TRE,including, but not limited to, another viral TRE.

[0146] It is understood that the present invention does not excludeadenovirus vectors containing additional genes under control of celltype-specific TREs. Accordingly, the invention provides adenoviralvectors comprising a third gene under transcriptional control of a thirdTRE. The third TRE may or may not be substantially identical to thefirst and/or second cell type-specific TREs, with all three TREsfunctional in the same cell. Preferably, the third gene is one thatcontributes to cytotoxicity (whether direct and/or indirect), morepreferably one that contributes to and/or enhances cell death,.Preferably the third TRE is cell type-specific. For example, anadenovirus vector may contain two PB-TREs and an hKLK2-TRE, or twoPSE-TREs and an hKLK2-TRE, each prostate cell specific and eachcontrolling the transcription of a different gene.

[0147] Accordingly, the invention provides adenoviral vectors comprisingat least an additional gene (beyond the first and the second genes)under transcriptional control of a cell type-specific TRE. Preferably,the additional gene is one that contributes to cytotoxicity (whetherdirect and/or indirect), more preferably 6ne that enhances cell death,.

[0148] Delivery of Adenoviral Vectors to Cells

[0149] The adenoviral vectors can be used in a variety of forms,including, but not limited to, naked polynucleotide (usually DNA)constructs; polynucleotide constructs complexed with agents tofacilitate entry into cells, such as cationic liposomes or othercompounds such as polylysine; packaged into infectious adenovirusparticles (which may render the adenoviral vector(s) more immunogenic);packaged into other particulate viral forms such as HSV or AAV;complexed with agents to enhance or dampen an immune response; complexedwith agents that facilitate in vivo transfection, such as DOTMA™,DOTAP™, and polyamines.

[0150] If an adenoviral vector is packaged into an adenovirus, theadenovirus itself may be selected to further enhance targeting. Forexample, adenovirus fibers mediate primary contact with cellularreceptor(s) aiding in tropism. See, e.g., Arnberg et al. (1997) Virol.227:239-244. If a particular subgenus of an adenovirus serotypedisplayed tropism for a target cell type and/or reduced affinity fornon-target cell types, such a subgenus (or subgenera) could be used tofurther increase cell-specificity of cytotoxicity and/or cytolysis.

[0151] The modified viruses may be delivered to the target cell in avariety of ways, depending upon whether the cells are in culture, exvivo or in vivo. For the prostate for the most part, the cells will bedelivered in vivo. Delivery can be achieved in a variety of ways,employing liposomes, direct injection, catheters, intravenousinhalation, topical applications, general transfection methods that arewell known in the art (such as calcium phosphate precipitation andelectroporation), and intravenous infusion, etc. Due to the highefficiency of transfection of adenoviruses, one can achieve a high levelof modified cells. In the case of neoplasia, where toxins are produced,the toxins will be released locally, so as to affect cells which may nothave been successfully transfected. In this manner, one can specificallyeliminate the neoplastic cells, without significant effect on the normalcells. In addition, expression of adenovirus proteins will serve toactivate the immune system against the target cells. Finally,proliferation of the adenovirus in a host cell will lead to cell death.The means of delivery will depend in large part on the particularadenoviral vector (including its form) as well as the type and locationof the target cells (i.e., whether the cells are in vitro or in vivo).

[0152] If used in a packaged adenovirus, the adenovirus may beadministered in an appropriate physiologically acceptable carrier at adose of about 10⁴ to 10¹¹. The multiplicity of infection will generallybe in the range of about 0.001 to 100. The viruses may be administeredone or more times, depending upon the immune response potential of thehost. If necessary, the immune response may be diminished by employing avariety of immunosuppressants, so as to permit repetitiveadministration, without a strong immune response.

[0153] If administered as a polynucleotide construct (i.e.; not packagedas a virus) about 0.01 μg to 1000 μg of an adenoviral vector can beadministered. The adenoviral vectors may be administered one or moretimes, depending upon the intended use and the immune response potentialof the host or may be administered as multiple simultaneous injections.If an immune response is undesirable, the immune response may bediminished by employing a variety of immunosuppressants, so as to permitrepetitive administration, without a strong immune response. If packagedas another viral form, such as HSV, the amount to be administered isbased on standard knowledge about that particular virus (which isreadily obtainable from, for example, published literature) and can bedetermined empirically.

[0154] In some embodiments, a packaged adenovirus vector(s) is complexedto a hydrophilic polymer to create a masked adenovirus. The hydrophilicpolymer is attached (covalently or non-covalently) to the capsidproteins of the adenovirus, particularly the hexon and fiber proteins.In preferred embodiments, the adenovirus vectors of the instantinvention are complexed with masking agents to create masked adenovirusvectors. Masked adenoviruses are advantageous due to (a) the masking ofthe adenovirus surface to adenovirus neutralizing antibodies or opsoninswhich are in circulation and (b) increasing systemic circulation time ofadenovirus particles by reduction of non-specific clearance mechanismsin the body (i.e. macrophages, etc.). In the in vivo context, thesystemic delivery of a masked adenovirus results in a longer circulationof virus particles, less immunogenicity, and increased biodistributionwith a decrease in clearance by the liver and spleen. Extensive researchhas been done on modification of proteins and lipids with hydrophilicpolymers (especially PEG) but the inventors are unaware of any other useof masking agents in conjunction with adenovirus or adenovirus vectors.

[0155] Accordingly, the invention provides an adenovirus complexed witha masking agent. Preferably the masking agent is PEG. A schematic of onemethod of making a masked adenovirus is depicted in FIG. 20 (also seeExample 8). A preferred embodiment is a masked adenovirus comprising anadenovirus vector(s) described herein with a more preferred embodimentcomprising a pegylated adenovirus comprising an adenovirus describedherein. The invention also provides methods to make and use these maskedadenoviruses, which are evident from the description herein.

[0156] The masking agents may be of various molecular weights, as longas the desired complex and requisite functionality is obtained. Mostmasking agents obtained from commercial sources are normallypolydisperse in relation to the state molecular weight (i.e. the maskingagent is supplied in a distribution of molecular weights surrounding thenominal molecular weight). Masking agents useful in masking adenovirusesaccording to the instant invention may have nominal weights of about2000 to about 50,000; preferably about 2500 to about 30,000; preferably,about 3000 to about 25,00, more preferably about 5000 to about 20,000.Preferably, the nominal molecular weight is less than about 20,000, morepreferably less than about 10,000, more preferably less than about 7500,more preferably less than about 5000. Preferably, the masking agents isPEG with a nominal molecular weight of less than about 5000 Da. Mixturesof different weight masking agents are also contemplated.

[0157] The masking may be covalently or non-covalently attached. In thecase of non-covalent attachment, the attachment may be electrostatic,hydrophobic, or affinity interactions. The masking agents used fornon-covalent attachment may be modified masking agents (i.e. the maskingagent is synthesized or modified to contain particular chemical moietiesnot normally present in the masking agent). Masking agents useful forelectrostatic attachment to adenoviral vectors will be masking agentswhich contain, or have been modified or synthesized to contain, chargedmoieties which bind to the adenovirus surface by electrostaticinteraction. Negatively charged masking agents include masking agentswhich contain phosphate groups, sulfate groups, carboxy groups, and thelike. Quaternary amine groups are useful as positively charged moietiesfor electrostatic, non-covalent attachment of masking agents toadenovirus. Masking agents containing or modified or synthesized tocontain hydrophobic groups, such as lipids (e.g.phosphatidylethanolamine and the like) and other hydrophobic groups(such as phenyl groups or long alkyl chains) can be complexed toadenoviral vectors by hydrophobic interaction with stable hydrophobicregions on the virus. Affinity masking agents can be made using anysmall molecule, peptide or protein which binds to adenovirus. Theaffinity and hydrophobic moieties may be attached to the masking agentby any method known in the art, preferably by chemical crosslinking witha chemical crosslinker.

[0158] If the masking agent is covalently attached, a chemicalcrosslinker is preferably used to covalently bond the masking agent tothe adenovirus. The crosslinker may be any crosslinker capable ofcreating a covalent linkage or bridge between the masking agent and theadenovirus. Direct crosslinking, in which the adenovirus masking agentand a separate crosslinker molecule are reacted, may be employed tocreate covalently masked adenovirus, using any chemical crosslinkerknown in the art which will create crosslinks between the masking agentand the protein. Either the masking agent or the adenovirus may bemodified prior to the crosslinking reaction, so that the chemicalcrosslinker will react with the two molecules (e.g. the masking agentmay be modified to add amine groups, allowing it to be crosslinked tothe adenovirus by crosslinking agents which react with amines.)

[0159] Preferably, either the masking agent or the adenovirus is firstactivated by reaction with a crosslinking agent. Unreacted crosslinkeris them removed from the masking agent or adenovirus. The activationpreferably results in one or two molecules of crosslinking agent permolecule of masking agent, more preferably a single molecule ofcrosslinking agent per molecule of masking agent. The activated maskingagent or adenovirus is then mixed with adenovirus, if the masking agentis activated, or masking agent, if the adenovirus is activated, underthe appropriate reaction conditions to form masked adenovirus.Preferably the masking agent is activated, then reacted with adenovirus.

[0160] The preferred masking agent is PEG. Preferred activated PEGsinclude, but are not limited to: nucleophilic crosslinking PEGs such asN terminal amine PEG, PEG amino acid esters, PEG hydrazinehydrochloride, thiol PEGs, and the like; carboxyl PEGs includingsuccinate PEG, carboxymethylated PEG, PEG-proprionic acid, and PEG aminoacids, sulfhydryl-selective PEGs such as PEG-maleimide,PEG-orthopyridyl-disulfide and the like, heterofunctional PEGs includingamines and acids PEG, NHS-maleimide PEG and NHS-vinylsulfone PEG; PEGsilanes; biotin PEGs; vinyl derivatives of PEG such as allyl PEG, PEGacrylate, PEG methacrylate, and the like, and electrophilic active PEGs,including PEG succinimidyl succinate, PEG succinimidyl succinamide, PEGsuccinimidyl proprionate, succinimidyl ester of carboxymethylated PEG,PEG2-NHS, succinimidyl esters of amino acid PEGs, pendant modified PEGMHS esters (such as those available from Innophase, Inc.), PEG-glycidylether (epoxide), PEG-oxycarbonylimidazole, PEG nitrophenyl carbonates,PEG trichlorophenyl carbonates, PEG treslate, PEG aldehyde, PEGisocyanate, copolymers of PEG allyl ether and maleic anhydride, PEGvinylsulfone, and other activated PEGs as will be apparent to one ofskill in the art. The activated PEG is preferably PEG-N-hydroxysuccinimidyl succinamide or PEG- succinimidyl succinate, more preferablyPEG-N-hydroxy succinimidyl succinamide.

[0161] Host Cells and Target Cells

[0162] The present invention also provides host cells and target cellscomprising (i.e., transformed with) the adenoviral vectors describedherein. Host cells include both prokaryotic and eukaryotic host cells aslong as sequence requisite for maintenance in that host, such asappropriate replication origin(s), are present. For convenience,selectable markers are also provided. Prokaryotic host include bacterialcells, for example, E. coli and mycobacteria. Among eukaryotic hostcells are yeast, insect, avian, amphibian, plant and mammalian hostcells. Host systems are known in the art and need not be described indetail herein.

[0163] Suitable target cells for the adenovirus vectors of the inventioninclude any eukaryotic cell type that allows function of the celltype-specific TREs, preferably mammalian, more preferably human, evenmore preferably neoplastic cells. Suitable target cells also include anycells that produce proteins and other factors necessary for expressionof the gene under control of the cell type-specific TREs, such factorsnecessary for said expression are produced naturally or recombinantly.For example, if the cell type-specific TRE(s) used is prostatecell-specific, the cells are preferably prostate cells, for exampleLNCaP cells. The prostate cells used may or may not be producing anandrogen receptor, depending on whether the promoter used isandrogen-inducible. If an androgen-inducible promoter is used,non-androgen receptor producing cells, such as HLF, HLE, and 3T3 and thenon-AR-producing prostate cancer cells PC3 and DU145 can be used,provided an androgen receptor-encoding expression vector is introducedinto the cells along with the adenovirus. If the cell type-specificTRE(s) used is derived from the AFP gene, for example, suitable hostcells include any cell type that produces AFP, including but not limitedto, Hep3B, HepG2, HuH7, HuH1/C12. Activity of a given TRE in a givencell can be assessed by measuring the level of expression of aoperably-linked reporter gene using standard assays. The comparison ofexpression between cells in which the TRE is suspected of beingfunctional and the control cell indicates the presence or absence oftranscriptional enhancement.

[0164] Comparisons between or among various TREs can be assessed bymeasuring and comparing levels of expression within a single target cellline. It is understood that absolute transcriptional activity of a TREwill depend on several factors, such as the nature of the target cell,delivery mode and form of a TRE, and the coding sequence that is to beselectively transcriptionally activated. To compensate for variousplasmid sizes used, activities can be expressed as relative activity permole of transfected plasmid. Alternatively, the level of transcription(i.e., mRNA) can be measured using standard Northern analysis andhybridization techniques. Levels of transfection (i.e., transfectionefficiencies) are measured by co-transfecting a plasmid encoding adifferent reporter gene under control of a different TRE, such as theCMV immediate early promoter. This analysis can also indicate negativeregulatory regions, i.e., silencers.

[0165] Compositions

[0166] The present invention also includes compositions, includingpharmaceutical compositions, containing the adenoviral vectors describedherein. Such compositions are useful for administration in vivo, forexample, when measuring the degree of transduction and/or effectivenessof cell killing in an individual. Preferably, these compositions furthercomprise a pharmaceutically acceptable excipient. These compositions,which can comprise an effective amount of an adenoviral vector of thisinvention in a pharmaceutically acceptable excipient, are suitable forsystemic administration to individuals in unit dosage forms, sterileparenteral solutions or suspensions, sterile non-parenteral solutions ororal solutions or suspensions, oil in water or water in oil emulsionsand the like. Formulations for parenteral and nonparenteral drugdelivery are known in the art and are set forth in Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing (1990).Compositions also include lyophilized and/or reconstituted forms of theadenoviral vectors (including those packaged as a virus, such asadenovirus) of the invention.

[0167] Other compositions are used, and are useful for, detectionmethods described herein. For these compositions, the adenoviral vectorusually is suspended in an appropriate solvent or solution, such as abuffer system. Such solvent systems are well known in the art.

[0168] Kits

[0169] The present invention also encompasses kits containing anadenoviral vector of this invention. These kits can be used fordiagnostic and/or monitoring purposes, preferably monitoring. Proceduresusing these kits can be performed by clinical laboratories, experimentallaboratories, medical practitioners, or private individuals. Kitsembodied by this invention allow one to detect the presence of targetcells in a suitable biological sample, such as biopsy specimens.

[0170] The kits of the invention comprise an adenoviral vector describedherein in suitable packaging. The kit may optionally provide additionalcomponents that are useful in the procedure, including, but not limitedto, buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, instructions, and interpretive information.

[0171] Preparation of the Adenovirus Vectors of the Invention

[0172] The adenovirus vectors of this invention can be prepared usingrecombinant techniques that are standard in the art. Generally, celltype-specific TREs are inserted 5′ to the adenoviral genes of interest,preferably one or more early genes (although late gene(s) may be used).Cell type-specific TREs can be prepared using oligonucleotide synthesis(if the sequence is known) or recombinant methods (such as PCR and/orrestriction enzymes). Convenient restriction sites, either in thenatural adeno-DNA sequence, or introduced by methods such as PCR orsite-directed mutagenesis, provide an insertion site for the celltype-specific TREs. Accordingly, convenient restriction sites forannealing (i.e., inserting) cell type-specific TREs can be engineeredonto the 5′ and 3′ ends of the cell type-specific TRE using standardrecombinant methods, such as PCR.

[0173] Polynucleotides used for making adenoviral vectors of thisinvention may be obtained using standard methods in the art such aschemical synthesis recombinant methods and/or obtained from biologicalsources.

[0174] The vectors are conveniently prepared by employing two plasmids,one plasmid providing for the left-hand region of adenovirus and theother plasmid providing for the right hand region, where the twoplasmids share at least about 500 nt of middle region for homologousrecombination. In this way, each plasmid, as desired, may beindependently manipulated, followed by cotransfection in a competenthost, providing complementing genes as appropriate, or the appropriatetranscription factors for initiation of transcription from the PSE forpropagation of the adenovirus.

[0175] For convenience, plasmids are available that provide thenecessary portions of the adenovirus. Plasmid pXC.1 (McKinnon (1982)Gene 19:33-42) contains the wild-type left-hand end of Ad5. pBHG10provides the right-hand end of Ad5, with a deletion in E3. The deletionin E3 provides room in the virus to insert the 2 kb minimal PSE withoutdeleting the wild-type enhancer-promoter. The gene for E3 is located onthe opposite strand from E4 (r-strand).

[0176] For manipulation of the early genes, the transcription start siteof Ad5 E1A is at nt 560 and the ATG start site of the E1A protein is atnt 610 in the virus genome. This region can be used for insertion of thecell specific element, e.g., PSE. Conveniently, a restriction site maybe introduced by employing the polymerase chain reaction (PCR), wherethe primer that is employed may be limited to the Ad5 genome, or mayinvolve a portion of the plasmid carrying the Ad5 genomic DNA. Forexample, where pBR322 is the backbone, the primers may use the EcoRIsite in the pBR322 backbone and the XpaI site at nt 1339 of Ad5. Bycarrying out the PCR in two steps, where overlapping primers at thecenter of the region introduce a sequence change resulting in a uniquerestriction site, one can provide for insertion of the cell specificresponse element at that site.

[0177] A similar strategy may also be used for insertion of the cellspecific response element to regulate E1B. The E1B promoter of Ad5consists of a single high-affinity recognition site for SpI and a TATAbox. This region extends from 1636 to 1701 nt. By insertion of the cellspecific response element in this region, one can provide for cellspecific transcription of the E1B gene. By employing the left-handregion modified with the cell specific response element regulating E1A,as the template for introducing the cell specific response element toregulate E1B, the resulting adenovirus will be dependent upon the cellspecific transcription factors for expression of both E1A and E1B.

[0178] For example, we have introduced an AgeI site 12 bp 5′ to the E1Ainitiation codon (Ad5 nucleotide 547) by oligo-directed mutagenesis andlinked PCR. In addition, an EagI site was created upstream of the E1Bstart site by inserting a G residue at Ad5 nt 1682 by oligonucleotidedirected mutagenesis. To simplify insertion of a TRE in the EagI site,the endogenous EagI site in CN95 was removed by digestion with EagI,treatment with mung bean nuclease, and religation to construct CN114. Inthis way, we generated an adenovirus vector containing unique AgeI andEagI sites in the proximal upstream region to E1A and E1B, respectively.Using these unique sites, one can insert a TRE which has engineered AgeIor EagI sites, thus simplifying construction of recombinant adenovirusvectors. Accordingly, the invention includes an adenoviral vectorcomprising a unique AgeI site 5′ of the E1A initiation codon and aunique EagI site 5′ of E1B.

[0179] For E4, one must use the right-hand portion of the adenovirusgenome. The E4 transcription start site is predominantly at nt 35605,the TATA box at nt 35631 and the first AUG/CUG of ORF1 is at nt 35532(Virtanen et al. (1984) J. Virol. 51:822-831). Using any of the abovestrategies for the other genes, the cell specific response element maybe introduced in the region between the transcription start site and theinitiation codon. Once again, by employing a previously manipulatedadenovirus genome, one can provide for a plurality of genes beingdependent upon the target cell specific transcription factor, insuringthat the adenovirus will be incapable of replication in cells lackingthese transcription factors.

[0180] Similarly, a cell type-specific TRE may be inserted upstream ofthe E2 gene to make its expression cell type-specific. The E2 earlypromoter, mapping in Ad5 from 27050-27150, consists of a major and aminor transcription initiation site, the latter accounting for about 5%of the E2 transcripts, two non-canonical TATA boxes, two E2Ftranscription factor binding sites and an ATF transcription factorbinding site (for a detailed review of the E2 promoter architecture seeSwaminathan et al., Curr. Topics in Microbiol. and Immunol. (1995) 199part 3:177-194).

[0181] The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable to geneticmanipulation. However, the E2 early promoter overlaps only for a fewbase pairs with sequences coding for a 33-kD protein on thecounterstrand. Notably, the SpeI restriction site (Ad5 position 27082)is part of the stop codon for the above mentioned 33 kD protein andconveniently separates the major E2 early transcription initiation siteand TATA-binding protein site from the upstream transcription factorbiding sites E2F and ATF. Therefore, insertion of a cell type-specificTRE having SpeI ends into the SpeI site in the plus-strand would disruptthe endogenous E2 early promoter of Ad5 and should allow TRE regulatedexpression of E2 transcripts.

[0182] For E4, one must use the right hand portion of the adenovirusgenome. The E4 transcription start site is predominantly at nt 35609,the TATA box at nt 35638 and the first AUG/CUG of ORF1 is at nt 35532.Virtanen et al. (1984) J. Virol. 51: 822-831. Using any of the abovestrategies for the other genes, a heterologous TRE may be introducedupstream from the transcription start site. For the construction ofmutants in the E4 region, the co-transfection and homologousrecombination are performed in W162 cells (Weinberg et al. (1983) Proc.Natl. Acad. Sci. USA 80:5383-5386) which provide E4 proteins in trans tocomplement defects in synthesis of these proteins.

[0183] Methods of packaging adenovirus polynucleotides into adenovirusparticles are known in the art and are described in the Examples.

[0184] Methods Using the Adenovirus Vectors of the Invention

[0185] The subject vectors can be used for a wide variety of purposes,which will vary with the desired or intended result. Accordingly, thepresent invention includes methods using the adenoviral vectorsdescribed above. In one embodiment, methods for using adenovirus vectorscomprise introducing an adenovirus vector into a cell, preferably aeukaryotic cell, more preferably a mammalian cell.

[0186] Purposes for introducing transient expression include indicationsthat may be treated involving undesired proliferation other than tumors,such as psoriatic lesions, restenosis, wound healing, tissue repair,enhanced immune response, resistance to infection, production offactors, enhanced proliferation, investigation of metabolic or otherphysiological pathways, comparison of activity of cells in the presenceand absence of the adenovirus introduced transgene, by comparing theactivity of the cell before, during and after the modification with theadenovirus, etc. The subject vectors can be used to free a mixture ofcells of a particular group of cells, where the group of cells is thetarget cells. By having the adenovirus be selectively competent forpropagation in the target cells, only those cells will be killed onproliferation of the adenovirus. By combining the adenovirus with themixture of cells, for example, in culture or in vivo, the adenoviruswill only be capable of proliferation in the target cells. In this waycells other than the target cells will not be affected by theadenovirus, while the target cells will be killed. The expansion of theadenovirus due to propagation in the target cells will ensure that themixture is substantially freed of the target cells. Once the targetcells are destroyed, the adenovirus will no longer be capable ofpropagation, but in culture may be retained so as to continually monitorthe mixture for recurrence of the target cell, e.g., a mutated cell orneoplastic cell.

[0187] By identifying genes that are expressed specifically by thetarget host cells, based on the nature of the cells, their level ofmaturity or their condition, the target cell specific response elementcan be used to provide genetic capability to such cells, where thegenetic capability will be absent in other cells, even when transfectedwith the adenovirus vehicle.

[0188] In one embodiment, methods for using adenovirus vectors compriseintroducing an adenovirus vector into a target cell, preferably aneoplastic cell. In another embodiment, methods for using adenovirusvectors comprise introducing an adenovirus vector into a prostate cell.In another embodiment, methods for using adenovirus vectors compriseintroducing an adenovirus vector into a liver cell. In anotherembodiment, methods for using adenovirus vectors comprise introducing anadenovirus vector into a breast cancer cell. In another embodiment,methods for using adenovirus vectors comprise introducing an adenovirusvector into a colon cancer cell.

[0189] In one embodiment, methods are provided for conferring selectivecytotoxicity in cells which allow function of the cell type-specificTRE, comprising contacting cells with an adenovirus vector describedherein, such that the adenovirus vector(s) enters, i.e., transduces thecell(s). Cytotoxicity can be measured using standard assays in the art,such as dye exclusion, ³H-thymidine incorporation, and/or lysis.

[0190] In another embodiment, methods are provided for propagating anadenovirus specific for cells which allow function of the celltype-specific TRE(s), preferably eukaryotic cells, more preferablymammalian cells. These methods entail combining an adenovirus vectorwith mammalian cells which allow function of the cell type-specificTREs, whereby said adenovirus is propagated.

[0191] Another embodiment provides methods of killing cells that allow acell type-specific TRE to function (i.e., target cells) comprisingcombining the mixture of cells with an adenovirus vector of the presentinvention. The mixture of cells is generally a mixture of cancerouscells in which the cell type-specific TREs are functional and normalcells, and can be an in vivo mixture or in vitro mixture.

[0192] The invention also includes methods for detecting cells in whicha cell type-specific TRE is functional in a biological sample. Thesemethods are particularly useful for monitoring the clinical and/orphysiological condition of an individual (i.e., mammal), whether in anexperimental or clinical setting. For these methods, cells of abiological sample are contacted with an adenovirus vector, andreplication of the adenoviral vector is detected. A suitable biologicalsample is one in which target cells may be or are suspected to bepresent. Generally, in mammals, a suitable clinical sample is one inwhich target cancerous cells are suspected to be present. Such cells canbe obtained, for example, by needle biopsy or other surgical procedure.Cells to be contacted may be treated to promote assay conditions such asselective enrichment and/or solubilization. In these methods, targetcells can be detected using in vitro assays that detect proliferation,which are standard in the art. Examples of such standard assays include,but are not limited to, burst assays (which measure virus yields) andplaque assays (which measure infectious particles per cell). Also,propagation can be detected by measuring specific adenoviral DNAreplication, which are also standard assays.

[0193] The invention also provides methods of modifying the genotype ofa target cell, comprising contacting the target cell with an adenovirusvector described herein, wherein the adenoviral vector enters the cell.

[0194] The invention further provides methods of suppressing tumor cellgrowth, comprising contacting a tumor cell with an adenoviral vector ofthe invention such that the adenoviral vector enters the tumor cell andexhibits selective cytotoxicity for the tumor cell. Tumor cell growthcan be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and stopping tumor growth, as wellas tumor shrinkage. “Suppressing” tumor growth indicates a growth statethat is curtailed when compared to growth without contact with, i.e.,transfection by, an adenoviral vector described herein. See Example 3,FIG. 6.

[0195] The invention also provides methods of lowering the levels of atumor cell marker in an individual, comprising administering to theindividual an adenoviral vector of the present invention, wherein theadenoviral vector is selectively cytotoxic in cells producing the tumorcell marker. Tumor cell markers include, but are not limited to, PSA,CEA and hK2. Methods of measuring the levels of a tumor cell marker areknown to those of ordinary skill in the art and include, but are notlimited to, immunological assays, such as enzyme-linked immunosorbentassay (ELISA), using antibodies specific for the tumor cell marker. Ingeneral, a biological sample is obtained from the individual to betested, and a suitable assay, such as an ELISA, is performed on thebiological sample. See Example 3, FIG. 7.

[0196] The invention also provides methods of treatment, in which aneffective amount of an adenoviral vector(s) described herein isadministered to an individual. For example, treatment using anadenoviral vector(s) in which at least one cell type-specific TRE isspecific for prostate cells (e.g., PSE-TRE, PB-TRE, and/or hKLK2-TRE) isindicated in individuals with prostate-associated diseases as describedabove, such as hyperplasia and cancer. In this example, also indicatedare individuals who are considered to be at risk for developingprostate-associated diseases, such as those who have had disease whichhas been resected and those who have had a family history ofprostate-associated diseases. Determination of suitability ofadministering adenoviral vector(s) of the invention will depend, interalia, on assessable clinical parameters such as serological indicationsand histological examination of tissue biopsies. Generally, apharmaceutical composition comprising an adenoviral vector(s) isadministered. Pharmaceutical compositions are described above.

[0197] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLE 1 Replication Competent Prostate-Specific AttenuatedAdenoviruses

[0198] Replication-competent adenoviral vectors were constructed inwhich a PSE mediates transcription of at least one adenoviral gene.

[0199] 1. Ad5 with PSE Driving Expression of E1A

[0200] The cloning and characterization of a minimal prostate-specificenhancer (PSE) is described in Prostate Specific Antigen Expression isRegulated by an upstream Enhancer (Schuur et al., supra). Plasmid CN71contains our minimal PSE (from−5322 bp to −3875bp relative to thetranscription start site of the PSA gene) and −532 to +11 of the PSApromoter. CN71 was cut with XhoI/HindIII which removes the PSA promoter.A shorter promoter, from −230 to +7, amplified by PCR using primers:18.119, 5′-GGACCTCGAGGTCTCCATGAGCTAC, and (SEQ ID NO:12) 15.59B,5′-AGCTCGAGCTTCGGGATCCTGAG. (SEQ ID NO:13)

[0201] The PCR product was cut with XhoI/HindIII and ligated back intoXhoI/HindIII cut CN71 creating CN105.

[0202] 1A. Attenuated Ad5 with PSE Driving E1A and Retaining theEndogenous Ad5 E1A Promoter and Enhancer

[0203] The E1A gene is expressed immediately after viral infection (0-2hours) and before any other viral genes. The E1A protein acts as atrans-acting, positive-acting transcriptional regulatory factor requiredfor the expression of the other early viral genes, E1B, E2, E3, E4, andthe promoter proximal genes of the major late genes. Despite thenomenclature, the promoter proximal genes driven by the major latepromoter are expressed during early times after Ad5 infection (Flint(1982) Biochem. Biophys. Acta 651:175-208; Flint (1986) Advances VirusResearch 31:169-228; Grand (1987) Biochem. J. 241:25-38). In the absenceof a functional E1A gene, viral infection does not proceed for the geneproducts necessary for viral DNA replication are not produced (Nevins(1989) Adv. Virus Res. 31:35-81). The transcription start site of Ad5E1A is at nt 560 and the ATG start site of the E1A protein is at nt 610in the virus genome.

[0204] pXC.1 was purchased from Microbix Biosystems Inc. (Toronto).pXC.1 contains Adenovirus 5 sequences from bp22 to 5790. We haveintroduced an AgeI site 12 bp 5′ to the E1A initiation codon (Ad5nucleotide 547) by oligo-directed mutagenesis and linked PCR. Theplasmid pXC.1 was PCR amplified using primers:

[0205] 15.133A, 5′-TCGTCTTCAAGAATTCTCA (SEQ ID NO: 14), containing anEcoRI site, and

[0206] 15.134B, 5′-TTTCAGTCACCGGTGTCGGA (SEQ ID NO: 15), containing anextra A to introduce an AgeI site. This created a segment from the EcoRIsite in the pBR322 backbone to Ad5 nt 560. A second segment of pXC.1from Ad nucleotide 541 to the XbaI site at Ad nucleotide 1339 wasamplified using primers:

[0207] 15.133B, 5′-GCATTCTCTAGACACAGGTG (SEQ ID NO: 16) containing anXbaI site, and

[0208] 15.134A, 5′-TCCGACACCGGGTGACCTGAAA (SEQ ID NO: 17), containing anextra T to introduce an AgeI site. A mixture of these two PCR amplifiedDNA segments was mixed and amplified with primers 3 and 4 to create aDNA segment from the EcoRI site to the XbaI site of pXC.1. This DNAsegment encompasses the leftmost 1317 bases of Adenovirus sequence andcontained an AgeI site at Ad nucleotide 547. This DNA segment was usedto replace the corresponding segment of pXC.1 to create CN95. Similarly,a PSE with AgeI ends was PCR amplified from CN105 using primers:

[0209] 15.176A, 5′-CATTAACCGGTACCTCTAGAAAATCTAGC (SEQ ID NO: 18) and

[0210] 15.176B, 5′-CATTAACCGGTAAGCTTGGGGCTGGGG (SEQ ID NO: 19) andcloned into CN95. The virus created by homologous recombination of CN96and BHG10 was designated CN706.

[0211] 1B. Attenuated Ad5 with PSE Driving Ad5 E1A Deleted for the Ad5Endogenous Promoter and Enhancer

[0212] In order to reduce ubiquitous expression of the E1A gene wedecided to delete the endogenous E1A transcription regulatory DNAsequences. The transcriptional regulatory sequences of the E1A gene areintricately embedded in DNA sequences essential for DNA packaging (seeGraeble and Hearing (1992) and References cited therein). Graeble andHearing (1990) have shown that an Adenovirus 5 with a deletion from bp194 to bp 316 which eliminates all transcriptional regulatory elementsand retains only three out of seven packaging signals reduced the yieldonly 3-fold compared to wild type. These observations suggested that theE1A transcription regulatory sequences are dispensable and the loss ofthe first three out of seven packaging signals allowed virus productionin acceptable quantities.

[0213] a. In the first variant, the region of the Ad5 genome containingthe E1A enhancer and promoter and the Ad5 packaging sequence weredeleted and replaced with a synthetic DNA segment containing a mutatedpackaging sequence and a PCR amplified segment of the PSE from CN127. Inthis construction the EcoRI/XbaI fragment of pXC.1 containing the first1339 bases of the Ad5 genome was cloned into pUC19 to construct CN172 asa substrate for further manipulations. The DNA sequences correspondingto Ad5 nt 123 to nt 497 were deleted from CN172 by PCR amplificationusing primers:

[0214] 26.153.1, 5′-CCGCTCGAGATCACACTCCGCCACAC (SEQ ID NO: 20)containing an XhoI site, and

[0215] 26.153.2, 5′-CCGCTCGAGCACTCTTGAGTGCCA (SEQ ID NO: 21), containingan XhoI site. Cleavage of the PCR product with XhoI followed byreligation resulted in CN178 in which an XhoI site replaced Ad5 nt 123to 497. The synthetic DNA segment containing the mutated Ad5 packagingsequences was composed of the following two strands: 26.160.1:5′-TCGAGGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGACTC(SEQ ID NO:22)TTCGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGG 26.160.2:5′-GATCCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACAAAATTATTCAG(SEQ ID NO:23)ATTTCGAAGAGTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAATTTACTACAACATCCC

[0216] The strands were annealed and kinased using T4 polynucleotidekinase to form the dsDNA and allow ligation to the other DNA segments inthe construct.

[0217] The PSE segment used for ligation was PCR amplified from CN127using primers:

[0218] 26.160.3, 5′-GGAAGATCTGAAATCTAGCTGATATAG (SEQ ID NO: 24),containing an XhoI site, and

[0219] 19.16.5,5′-TTCTCGAGAAGCTTGGGGCTGGGG (SEQ ID NO: 25), containingXhoI and HinDIII sites. For ligation, the PSE PCR product and CN178 wereboth cleaved with XhoI. The XhoI cut CN178, XhoI cut PSE PCR product,and the kinased packaging oligonucleotide were mixed in equal molarratios and ligated with T4 DNA ligase. The resulting recombinant wasdesignated CN201. The EcoRI/XbaI segment of CN201 containing the mutatedpackaging sequence and PSE driving E1A was excised from CN201 and usedto replace the homologous segment of pXC.1 to generate CN202.

[0220] b. In the second variant, a different strategy was employed. Inorder to perform the deletion mutagenesis with a relatively smallplasmid, a 2297 bp EcoRI-XhoI fragment of plasmid CN145, which containsthe left end Adeno sequences including the E1A promoter region and thePSA enhancer, was subcloned into similarly cut pBluescript SKII+yielding plasmid CN169.

[0221] The plan for the deletion mutagenesis was to delete the sequencesfrom Ad position 194-301 and replace them with a SaII restriction site5′-GTCGAC-3′ which served as diagnostic marker to distinguishmutagenized plasmids from parental plasmids. The deletion eliminated allE1A core and E2F transcription regulatory elements as well as packagingsignals AI and AII, but will preserve packaging signals AIII, AIV, AV,AVI and AVII. To this end, two oligonucleotide primers were synthesized:

[0222] 28.134A, 5′-GTCGACGTGAAATCTGAATAATTTTGTGTTACTCATAGC (SEQ ID NO:26). This primer matches to sequences 302-334 in Ad5.

[0223]28.134B, 5′-CACCGGCGCACACCAAAAACGTC (SEQ ID NO: 27). This primermatches to sequences 171-193 in Ad5.

[0224] The PCR mutagenesis kit from Stratagene was used in the followingmanipulations. In a PCR tube, 15 pMol of each primer was added to 0.5pMol CN169; 1 mM dNTP, 2.5 μl 10×PCR 11 (Stratagene), dH₂O to 24 μl and0.5 μl each of Taq Polymerase and TaqExtender (Stratagene). The mixturewas overlaid with 20 μl mineral oil and programmed for PCR: 94° C. 4minutes, 63° C. 1 minute, 72° C. 4 minutes for cycle and 94° C. 1minute, 63° C. 1 minute, 72° C. 4 minutes for 10 cycles. l μl Dpn Irestriction enzyme (Stratagene) was added to cut parental DNA andincubated at 37° C. for 80 minutes followed by the addition of 1 μl PfuPolymerase (Stratagene) and incubation at 72° C. for 50 minutes to fillup protruding DNA ends generated during the former PCR process by theTaq polymerase. The PCR yielded a 5 kb linear DNA which was ligated withT4 DNA ligase to recircularize. XL-1 bacteria were transformed with theligation reaction and mutagenized recombinants were identified by virtueof the presence of the unique SaII restriction site. One of therecombinants, CN 179, was used to rebuild the parental plasmid CN145with the deletion by swapping the EcoRI-XhoI fragment of CN145containing the Adeno-and PSE sequences with the one of CN179, yieldingplasmid CN185. Plasmid CN185 was used in cotransfections with BHG11 intohuman 293 cells to generate recombinant Adenoviruses. Nine virus plaqueswere isolated. One virus isolate was designated CN724.

[0225] 2. Attenuated Ad5 with PSE Driving Expression of E1B

[0226] The E1B protein functions in trans and is necessary for transportof late mRNA from the nucleus to the cytoplasm. Defects in E1Bexpression also results in poor expression of late viral proteins and aninability to shut off host-cell protein synthesis. The promoter of E1Bhas been implicated as the defining element of difference in the hostrange of Ad40 and Ad5: clinically Ad40 is an enterovirus, whereas Ad5causes acute conjunctivitis (Bailey, Mackay et al. (1993) Virology193:631; Bailey et al. (1994) ibid 202:695-706). The E1B promoter of Ad5consists of a single high-affinity recognition site for SpI and a TATAbox.

[0227] To insert a PSE driving expression of E1B in Ad5, an EagI sitewas created upstream of the E1B start site by inserting a G residue atAd5 nt 1682 by oligonucleotide directed mutagenesis as above. Tosimplify insertion of the PSE in the EagI site the endogenous EagI sitein CN95 was removed by digestion with EagI, treatment with mung beannuclease, and religation to construct CN114. The primers:

[0228] 15.133A, 5′-TCGTCTTCAAGAATTCTCA (SEQ ID NO: 14), containing anEcoRI site, and

[0229] 9.42, 5′-GCCCACGGCCGCATTATATAC (SEQ ID NO: 28), containing anextra C, were used to amplify the segment between the EcoRI site and Ad5nt 1682.

[0230] Primers:

[0231] 9.39, 5′-GTATATAATGCGGCCGTGGGC (SEQ ID NO: 29) containing anextra G, and

[0232] 24.020, 5′-CCAGAAAATCCAGCAGGTACC (SEQ ID NO: 30), containing aKpnI site, were used to amplify the segment between 1682 and the KpnIsite at Ad5 nt 2048. Co-amplification the two segments with primers 9and 12 yields a fragment with an EagI site at Ad5 nt 1682 which was usedto replace the corresponding EcoRI/KpnI site in pXC.1 to constructCN124. PSE amplified from CN105 with primers:

[0233] 26.1.1, 5′-TAACGGCCGTCTAGAAATCTAGCTGA (SEQ ID NO: 31) and

[0234] 26.1.2, 5′-TAACGGCCGAAGCTTGGGCTGGG (SEQ ID NO: 32), with EagIends, was ligated into the EagI site of CN124 to construct CN125. Theresultant virus from homologous recombination of CN125 and BHG10 wasdesignated CN711.

[0235] 3. Attenuated Ad5 with PSE Driving Expression of Both E1A and E1B

[0236] A left end Ad5 plasmid with the PSE driving expression of bothE1A and E1B was constructed by PCR amplifying CN95 with primers 9-12 asdescribed for the construction of CN124. The resulting DNA segmentcontains the AgeI site derived from CN95 and the EagI site derived fromthe PCR mutagenesis. This DNA segment was cloned back into CN114 (theplasmid from which the EagI site was removed from pXC.1) to constructthe plasmid CN144. CN144 contains a single AgeI site at Ad5 nt 547, anda single EagI site at Ad5 nt 1682. PSE segments were PCR-amplified withAgeI ends from CN105 or EagI ends, also by PCR from CN105, as describedabove and ligated into the appropriate sites of CN144 to constructCN145. CN145 is a plasmid in which the PSE drives expression of both E1Aand E1B while retaining the Ad5 endogenous promoters and enhancers ofboth genes. Clones with the PSE in the left to right orientation werechosen. The endogenous Ad5 E1A and E1B promoter/enhancers were movedupstream by insertion of both PSE segments. The resultant virus derivedby homologous recombination of CN145 and BHG10 was designated CN716.

[0237] 4. Attenuated Ad5 with PSE Driving Expression of E4

[0238] E4 is located at the far right-hand end of the Ad5 genome andread right-to left from the 1-stand (Flint, supra). E4 can be deletedfrom the Ad5 genome and supplied in trans by W162 cells, a derivative ofVERO cells (Weinberg and Ketner, supra). The transcription products ofE4 are complex. Open-reading frames (ORF) 3 and ORF 6 of the E4transcription unit increase the accumulation of major late transcriptionunit mRNAs by binding the 55-kDa protein from E1B (Dix and Leppard(1993) J. Virol. 67:3226-3231) and heterodimers of E2F-1 and DP-1 (Helinand Harlow (1994) J. Virol. 68:5027-5035). Mutations such that neitherORF 3 nor ORF 6 encode functional proteins, produce plaques with anefficiency less than 10⁻⁶ that of wild-type virus (Bridge and Ketner(1989) J. Virol. 67:5911-5921).

[0239] To facilitate insertion of the PSE driving E4 expression, the 10kb EcoRI fragment of BHG10 containing the 3′ 8 kb of Ad5 plus a portionof the pBR322 backbone was cloned into the EcoRI site of BluescriptKSII+ to construct CN108. A DraII site at Ad nt 33906 was eliminated bypartial digestion of CN108, end filling with Klenow, and relegation toconstruct-CN113. An XhoI site was introduced at Ad nt 35577 byoligonucleotide directed mutagenesis and linked PCR as described aboveusing primers:

[0240] 10.1, 5′-TAACTCACGTTGTGCATTGT (SEQ ID NO: 33), containing a Drallsite,

[0241] 10.4, 5′-GGTGCCGTGCTCGAGTGGTGT (SEQ ID NO: 34), containing anextra C,

[0242] 10.3, 5′-ACACCACTCGAGCACGGCACC (SEQ ID NO: 35), containing anextra G,

[0243] 19.158, 5′-GCTACTATTCGACAGTTTGTACTG (SEQ ID NO: 36), containing aClal site.

[0244] The PCR product containing an XhoI site as well as DraIII andClal ends was used to replace the corresponding DraIII/Clal fragment ofCN113 to construct CN122.

[0245] Plasmid CN70 contains the minimal PSE (from−5322 bp to −4023 bprelative to the transcription start site of the PSA gene) and −532 to+11 of the PSA promoter. CN70 was cut with XhoI/HindIII which removesthe PSA promoter. A shorter promoter, from −230 to +7, amplified by PCRusing primers:

[0246] 18.119, 5′-GGACCTCGAGGTCTCCATGAGC TAC (SEQ ID NO: 12), and

[0247] 15.59B, 5′-AGCTCGAGCTTCGGGATCCTGAG (SEQ ID NO: 13), was ligatedin it's place to construct CN104. CN127 was constructed from CN104 asfollows: CN104 was cut with XhoI, end-filled with Klenow, and relegatedto remove the XhoI site. The PSE from CN127 was PCR amplified usingprimers:

[0248] 19.16.1, 5′-GGGTCGACGTACCTCTAGAAATCTAGC (SEQ ID NO: 37) and

[0249] 19.16.5, 5′-TTGTCGACMGCTTGGGGCTGGGG (SEQ ID NO: 25), to createSaII ends. This DNA segment was then ligated to XhoI cut CN122 to insertthe PSE in the correct orientation upstream of E4. The resulting plasmidwas designated CN135. The kanamycin resistance gene from pABS4(Microbix) was inserted into CN135 at the Pacl site to construct CN146;the EcoRI fragment of CN146 (containing the adenovirus sequences withthe inserted PSE and kanamycin resistance gene) was then ligated to thelarge EcoRI fragment of BHG10, replacing the homologous wild type Adsequences in BHG10. Recombinants were identified by resistance to bothampicillin and kanamycin, then the kanamycin gene was excised by PacIdigestion and relegation to yield CN190 which is BHG10 with the PSEinserted upstream of the E4 coding region.

[0250] 5. Attenuated Ad5 with PSE Driving Ad5 E1A containing CytosineDeaminase in ΔE3

[0251] A prostate specific adenovirus vector that contains the cytosinedeaminase (“cd”) gene incorporated into its genome could deliver thisgene to targeted tissue (i.e. prostate tumors). Consequently, infectedcancer cells would metabolize 5-FC and release the chemotherapeuticagent 5-FU into the surrounding tissue suppressing cell division, andexhibit the so-called “bystander effect” (Hirshowitz et al. (1995) HumanGene Ther. 6:1055-1063; Griffith and Jarvis (1993) J. Biol. Chem.268:20085-20090). In contrast, noninfected, nonproximal cells would notbe affected. This suggests two uses for the cd gene in an attenuatedadenovirus vector. First, cd can serve as an additional therapeuticagent to provide a bystander killing ability and expedite local tumorreduction without systemic toxicity (Moolten and Wells (1990) J. Nat'lCancer Inst. 82:297-300). Second, the gene can serve as a recallmechanism to halt a runaway infection by preventing viral DNA and RNAsynthesis in infected and noninfected, local cells.

[0252] The enzyme cytosine deaminase, which deaminates cytosine touracil, is found in many bacteria and fungi. These microorganisms canconvert 5-fluorocytosine (5-FC), a harmless prodrug, to 5-fluorouracil(5-FU), a highly toxic compound that inhibits both DNA and RNA synthesis(Calibrisi and Chabner Goodman and Gilman's The Pharmacological Basis ofTherapeutics (Eds. A. G. Gilman, T. Rall, A. S. Nies, and P. Taylor,Pergamon, N.Y.) (1990) 8 ed., pp 1209-1263); Damon et al. (1989).Because mammalian cells do not express significant amounts of the cdgene, they are not sensitive to 5-FU. Mammalian cells modified by genetransfer to express the gene can metabolize 5-FC, however. In thisapplication, cd acts as a “suicide gene” selectively conferringsensitivity to those cells that contain the gene.

[0253] Adenovirus Vector Construction. The plasmid pCMV-cd, whichcontains cd coding region downstream of the CMV promoter, was obtainedfrom David Crooks (Stanford). A SpeI restriction endonuclease sitelocated in a multiple cloning region between the promoter and the cd ATGwas removed by digesting the plasmid with enzymes which recognizesequences flanking the SpeI site, BamHI and EcoRI, filling the ends withKlenow, and relegating (CN130). With this site removed, the CMV-cdcassette was cloned by digesting CN130 with SpeI and ligating theappropriate fragment into the XbaI site in pABS4 (Microbix, Toronto), ashuttle plasmid containing the kanamycin-resistance gene (CN131). Bydigesting CN131 with PacI, a fragment containing the Kan^(R) gene andthe cd gene was isolated and ligated into similarly cut BHG11(Microbix), which contains a unique PacI site engineered in the E3region of Ad5 (CN141). The kan^(R) gene was removed by digesting CN141with SwaI and religating the vector (CN148).

[0254] Two Ad5 recombinant viruses containing the cd gene in the E3region were constructed. The first contains only the CMV-cd cassette inthe E3 region (CN719). The second has the CMV-cd cassette in E3 and theprostate specific enhancer (PSE) minimal element modulating expressionof E1A proteins (CN720). Viruses were generated by homologousrecombination in low passage 293 cells, a human kidney cell line thatexpresses Ad E1A and E1B proteins, accomplished by cotransfecting themwith pXCI/CN148 and CN145(PSE-E1A)/CN148.

[0255] In vitro Characterization. In this first functional assay, CN720,an attenuated, prostate-specific adenovirus containing the cd gene inthe E3 region, was studied to test its ability to confer 5-FCsensitivity on infected cells and neighboring cells. Wild type Ad5(CN702) was also tested. CV1 cells, a semipermissive monkey kidney cellline, seeded in four, 96 well microtitre plates in DMEM, 5% FBS, wereinfected in a series of 1:2 dilutions from wells 1-11 with either CN702or CN720. The multiplicity of infection of well one was approximatelytwenty-five for CN702 and two for CN720. Row 12 in each plate was leftas an noninfected control. One day post infection the media was changed.Two plates of cells, one infected with CN720 and one infected withCN702, were treated with 5 mM 5-FC. The media on the remaining twoplates was changed with complete DMEM only. These infected, untreatedcells illustrate the lytic ability of the virus and were used todifferentiate between the two causes of cell death in this experiment,virus cell lysis and 5-FU toxicity. The cells were fixed with 50%methanol-50% acetone and stained with Giemsa stain 6 days after theprodrug was administered. Plates were assayed by measuring absorbance at530 nm in a SpectraMAX 340 microtitre plate reader (Molecular Devices).Cell survival was calculated by relating the absorbance of the cells inthe noninfected wells to the absorbance in infected wells. The resultswere graphed as cell survival versus virus dilution.

[0256] Several conclusions can be made from this experiment. Mostimportant, the graph suggests that the recombinant adenoviruses areexpressing the cd gene. While the cell killing ability of both virusesappears to increase in the presence of 5-FC, perhaps due to ageneralized toxicity to high concentrations of the prodrug, the changein cell killing is dramatic for CN720. The graph of CN720 shows a clearcell survival difference between 5-FC treated cells and untreated cellsindicative of a 5-FU bystander effect. This result illustrates thepotential to exploit cd function to either enhance the killing potentialof Ad5 or to harness a runaway infection by generating an intracellularpool of toxic drug in noninfected cells that prevents DNA replication, arecall mechanism.

[0257] As an in vitro model, six 96 well plates were seeded with a humanintestine epithelia cell line, DLD-1, that is permissive to human Ad inDMEM, 10% FBS. They were infected as described above with Ad5-cd virus(CN719). Prodrug (1 mM) was added to one plate at each time point, 0hrs, 24 hrs, and 48 hrs post infection. The remaining three plates wereuntreated and served as infected controls. One set of two plates, onewith prodrug, one without, was harvested on day 7, 8, and 9 postinfection.

[0258] These results corroborate the previous data and extend it.Increased cell death is seen at all time points in infected pro-drugtreated cells relative to infected but untreated cells. These data alsoreveal that the bystander effect is more pronounced as the infectionbecomes more advanced. When 5-FC is added at 24 hours and at 48 hourspost infection, cell death is greater than when the prodrug is addedimmediately after initial infection. These data demonstrate that atissue specific adenovirus harboring the cd gene has superior killingability to wild type adenovirus.

[0259] 6. Attenuated Ad5 with PSE Driving E1A and SV40 T Antigen in ΔE3to Increase Host Range to Include Monkey and Human Cells

[0260] Human adenovirus does not efficiently replicate in monkey cells.Associated with decreased levels of fiber mRNA in the cytoplasm, theabortive infection is caused by defects in the late gene expressionregulated by E4 proteins (Ross and Ziff (1992) J. Virology66:3110-3117). Adenovirus-SV40 hybrids—shown to contain a small portionof the SV40 genome coding for the large T antigen integrated into the E3region of the adenovirus 2 genome, overcome this defect and lyse monkeycells (Lewis and Rowe (1970) ibid 5:413-420; Lewis et al, (1973) ibid11:655-664). The large T antigen (Tag) is believed to confer thishost-range capability on these hybrids (Tijan et al., (1979) PNAS75:1279-1283). Several Ad2-SV40 hybrids have been isolated from SV40 andAd2 infected cultures, each containing a conserved amount of the Tagcarboxy terminal coding region and varying lengths of amino terminalcoding region.

[0261] We have adopted this paradigm to develop Ad5 tissue specific,host-range mutants for use in monkey studies. Two strategies wereundertaken. The first used the host-range mutant Ad2+ND1, which harborsSV40 Tag coding sequence from map units 0.28-0.11, as a model (Zain &Roberts (1978) J. Mol. Biol 120:13). A 666 base pair PstI/BamHIrestriction fragment in the plasmid pDIS (obtained from EdgarSchrieber), a plasmid which contains the entire Tag coding sequence, theendogenous SV40 early promoter, and an inverted SV40 enhancer, containsthe appropriate 3′ sequence and was cloned via the shuttle plasmid pABS4(Microbix) into the unique PacI restriction site in the E3 region ofBHG11 (Microbix). Upstream of the coding sequence was cloned an oligo(+) strand:

[0262] 26.99.1, 5′-GTTIGTGTATITTTAGATCAAAGATGCTGCA (SEQ ID NO: 38), and(−) strand:

[0263] 26.99.2, 5′-GCATCTTTGATCTAAAATACACAAAC (SEQ ID NO: 39), thatcontains a splicing acceptor sequence, ribosome recognition sequences,and an ATG to achieve expression of the appropriate peptide (CN170).Expression of this construct is dependent on a transcript originatingfrom the major late promoter.

[0264] The second strategy involved creating an internal deletion in theTag sequence in the plasmid pDIS between the EcoNI site in the aminoterminal region and the PstI site in the carboxy terminal codingsequence by using an adapter oligo (+) strand:

[0265] 27.183.1, 5′-TAAAGGAGGAGATCTGCCTAAAACACTGCA (SEQ ID NO: 40), and(−) strand:

[0266] 27.183.2, 5′-GTGTTTTAGGCAGATCTCCTCCTTT (SEQ ID NO: 41).

[0267] The entire transcription unit, including the enhancer, promoter,and the coding sequence was excised by HpaII/BamHI digestion and clonedvia shuttle plasmid into the unique PacI site of BHG11 (CN183). Thismethod generates a discrete transcription unit in Ad5 sequence whoseexpression is not dependent on the major late promoter.

[0268] Two host-range Ad5-SV40 viruses were produced. Both contain thecarboxy termini of the Tag but lack the promoter. One is atissue-specific, attenuated virus with the prostate specific enhancer(PSE) modulating expression of the E1A proteins (CN725). The other iswild type Ad5 with a Tag insertion (CN726). Both were generated byhomologous recombination by cotransfecting 293 cells, a human kidneycell line that expresses Ad E1A and E1B proteins, with CN145(PSE-E1A) orpXCI (wild type Ad5 left hand end) and CN170.

[0269] Host-Range Mutant Characterization. Wild type Ad5 (CN702) andCN726 were plaqued on both 293 cells and CV1 cells, an African GreenMonkey kidney cell line. Plaques were counted in both cell monolayersand a ratio between the plaques in the two cell lines was determined.The ratio for CN726 and CN702 was 0.01 and 0.0007, respectively. Thecapability of replication of adenovirus in monkey cells allowspreclinical evaluation of recombinant attenuated adenoviruses inmonkeys, yielding valuable information for dosage and formulation ofthese viruses as therapeutic agents in humans.

[0270] 7. Construction of Recombinant DNA to Introduce Mutations in E2,the DNA Binding Protein (DBP), for the Generation of Recombinant Ad5with Extended Host Range Allowing Replication in Human and Monkey Cells

[0271] Wild type adenovirus type 5 is only replication competent inhuman cells. For preclinical evaluation of therapeutic attenuatedadenoviruses it would be desirable to test efficacy and toxicity inlarge human-like animals such as monkeys. A host range mutant hr404 hasbeen described that confers a replication phenotype of human Ad5 inmonkey cells (Klessig & Grodzicker (1979) Cell 17:957-966). The natureof the hr404 mutation was shown to be a single point mutation C—>T atadeno position 32657 in the DBP gene resulting in a change of Histidineto Tyrosine amino acid at codon 130 (HI30Y) in the 72K DNA bindingprotein (Kruijer et al. (1981) Nucleic Acids Res. 9:4439-4457).

[0272] We constructed a recombinant DNA molecule with the 5.8 kbEcoRI-BamHI fragment from plasmid BHG10 (Bett et al., supra) containingthe right end sequences of Adenovirus type 5 and introduced bysite-directed mutagenesis the H130Y mutation in the DBP gene. Thisplasmid should allow the construction of recombinant adenoviruses whichare capable to replicate in human and monkey cells.

[0273] The 5769 bp EcoRI-BamHI fragment of BHG10 (Bett et al., supra)was cloned into similarly cut pBluescript KSII+ resulting in plasmidCN184. In order to eliminate disturbing restriction sites, a 2568 bpXhoI fragment was deleted yielding plasmid CN 186. The mutagenesis upperPCR primer reads: 28.180U,5′-GCAACCCACCGGTGCTAATCAAGTATGGCAAAGGAGTAAGCGC-3 (SEQ ID NO:42)

[0274] The mutated T residue causing the H130Y mutation is shown in boldunderlined style. Shown in italics is the unique SgrAI site in pCN186.The lower PCR primer reads:

[0275] 28.180L, 5′-TGGCCTTGCTAGACTGCTCCTTCAGC-3′ (SEQ ID NO: 43)

[0276] PCR amplification was done with 100 pMol of each of theseprimers, 200 ng CN186 as template, 1 mM dNTP, 1×Pfu buffer (Stratagene),dH2O to 100

I, and 5U cloned Pfu polymerase (Stratagene) at 94° C. 1 minute, 60° C.1 minute, 72° C. 2 minutes for 30 cycles. The PCR yielded the expectedDNA fragment of 588 bp. The DNA fragment was purified with a Wizard DNAclean-up column (Promega) and digested with restriction enzymes SgrAIand AflII. The 473 bp fragment of interest containing the H130Y mutationwas gel purified and isolated. For reinsertion into the DBP gene, themutated DNA fragment was ligated with the 1639 bp AscI-SgrAl fragmentfrom CN184 and the 6609 bp AflII-AscI fragment from CN184 resulting inplasmid CN188.

[0277] Recombinant adenovirus genomes were constructed by in vitroligation of the 5.8 kb EcoRI-BamHI fragment of CN188 with a 21562 bpEcoRI-Bst1107 center DNA fragment of BHG10 and Bst1107-cut plasmidCN144. The resultant virus was designated CN723.

[0278] The capability of replication of adenovirus in monkey cellsallows preclinical evaluation of recombinant attenuated adenoviruses inmonkeys, yielding valuable information for dosage and formulation ofthese viruses as therapeutic agents in humans. Further, with the use ofthe hr404 mutation in CN723, the same virus used for monkey studies canbe used as the human clinical trial virus.

[0279] 8. Deletion of ORF 1,2,3 and Part of ORF 4 from the E4 Region ofAdenovirus Type 5

[0280] The E4 region codes for two polypeptides which are responsiblefor stimulating the replication of viral genomic DNA and for stimulatinglate gene expression. The protein products of open reading frames (ORFS)3 and 6 can both perform these functions, however the ORF 6 proteinrequires interaction with the E1B 55K protein for activity while the ORF3 protein does not. To further restrict viral replication to prostateepithelial cells E4 orfs 1-3 can be deleted, making viral DNAreplication and late gene synthesis dependent on E4 ORF 6 protein. Bycombining such a mutant with sequences in which the E1B region isregulated by the PSE, a virus can be obtained in which both the E1Bfunction and E4 function are dependent on the PSE driving E1B.

[0281] A virus of this type was constructed by combining sequences fromthe plasmid d11006 which contains an E4 deletion of ORFS 1-3 (Bridge &Ketner, J. Virol. (1989) 63:631-638) with BHG10, followed byco-transfection with CN144 to construct a recombinant virus. The plasmidpd11006 is cleaved with AvrII and AgeI to isolate sequences containingthe mutated E4 region. This DNA segment is used to replace thehomologous segment of CN108 cleaved with the same enzymes.

[0282] CN108 contains the 6 kb EcoRI fragment from BHG10 cloned intoBSKSII+. Due to the E3 deletion in BHG10, the AvrII site at Ad5 nt 28752had been deleted. AvrII still cut CN108 at Ad5 nt 35463; AgeI cut CN108at Ad5 nt 31102. The 4.4 kb AvrII/AgeI fragment from CN108 was replacedwith the 3.8 kb AvrII/AgeI fragment from d11006 producing CN203containing the E4 deletion. The EcoRI fragment from CN203 was clonedinto BHG10 to construct CN204. Homologous recombination of CN204 andCN144 yielded the virus CN726.

[0283] A similar virus of this type was constructed in the followingmanner. As previously described AvrII cut CN108 at Ad5 nt 35463. SapIcut CN108 twice, with one of the sites at Ad5 nt 34319. A complete AvrIIcut and a partial SapI cut of CN108 and religation removed 1144 bp fromE4 yielded CN205. The 5.3 kb EcoRI/BamHI fragment from CN205 was clonedinto similarly cut CN188 yielding CN206. The 14 kb BamHI fragment ofCN206 containing both the E4 deletion and the hr404 mutation was clonedin BamHI cut BHG10 producing CN207. Homologous recombination of CN144and CN207 in 293 cells yielded CN727.

[0284]9. PSE Controlling the E2 Region of Ad5

[0285] The E2 region of Adenovirus 5 codes for proteins related toreplication of the adenoviral genome, including the 72 kDa DNA-bindingprotein, the 80 kDa precursor terminal protein and the viral DNApolymerase. The objective is to control expression of the E2 genes bythe prostate-specific PSA enhancer/promoter in a recombinant adenovirus.

[0286] The E2 region of Ad5 is transcribed in a rightward orientationfrom two promoters, termed E2 early and E2 late, mapping at 76.0 and72.0 map units, respectively. While the E2 late promoter is transientlyactive during late stages of infection and is independent of the E1Atransactivator protein, the E2 early promoter is crucial during theearly phases of viral replication.

[0287] The E2 early promoter, mapping in Ad5 from nt 27053-27121consists of a major and a minor transcription initiation site, thelatter accounting for about 5% of the E2 transcripts, two non-canonicalTATA boxes, two E2F transcription factor binding sites and an ATFtranscription factor binding site (for a detailed review of the E2promoter architecture see Swaminathan & Thimmapaya, Current Topics inMicrobiology and Immunology (1995) 199 part 3:177-194.

[0288] The E2 late promoter overlaps with the coding sequences of the L4gene encoded by the counterstrand and is therefore not amenable forgenetic manipulation. However, the E2 early promoter overlaps only for afew base pairs with sequences coding for a 33 k protein on thecounterstrand. Notably, the SpeI restriction site (Ad5 position 27082)is part of the stop codon for the above mentioned 33 kDa protein andconveniently separates the major E2 early transcription initiation siteand TATA-binding protein site from the upstream transcription factorbiding sites E2F and ATF. Therefore, an insertion of the PSAenhancer/promoter into the SpeI site would disrupt the endogenous E2early promoter of Ad5 and should allow prostate-restricted expression ofE2 transcripts.

[0289] Construction of recombinant Ad5 with the PSA enhancer/promoter inthe E2 early promoter region. The BamHI-EcoRI fragment of Ad5 (positions21562-27331) encompassing the E2 region was previously subcloned intopBluescript KSII+ resulting in plasmid CN184. A variant of this plasmid,CN188, carrying a mutation in the DBP gene (HI30Y) allowing extendedhost range applications has been constructed and described above.

[0290] Plasmid CN188 was used for insertion of the PSA enhancer/promoterinto the E2 region. The plasmid was linearized with SpeI and the 540protruding ends were dephosphorylated with calf intestine alkalinephosphatase and then end-filled with Klenow polymerase and dNTP. Theblunt ended PSE enhancer/promoter was ligated to SpeI linearized, bluntended vector CN188. Recombinant DNAs with the PSE enhancer/promoterinthe appropriate orientation for directing transcription initiation intothe E2 region were identified. Plasmid CN196 contains the PSEenhancer/promoter in the backbone of CN188. The 5.3 kb EcoRI fragment ofplasmid CN205, containing a deletion of the orf 1, 2, 3 and 4 of the E4gene, was inserted in the appropriate orientation into EcoRI cut CN196,yielding plasmid CN197.

[0291] A recombinant viral genome with the PSE enhancer/promotercontrolling expression of the E1A, E1B and the E2 early genes and thehr404 mutation H130Y in the DBP gene and deletion of open reading frames1, 2, 3, and 4 of the E4 gene was obtained by in vitro ligation of the9152 bp BamHI-Bst11071 fragment of CN144 with the 15802 bpBst11071-BamHI fragment of BHG10 and the 12425 bp BamHI fragment ofCN197.

[0292] Virus Preparation

[0293] Viruses were prepared as described previously (above). Table 1,below, lists the combinations of right end and left end Ad5 plasmidsused to generate recombinant Ad5 with the desired features: TABLE 1Right End Virus Name Left End Plasmid Plasmid PSE-E1A CN704-708 CN96BHG10 PSE-E1A CN718 CN145 BHG10 PSE-E1B CN711 CN125 BHG111 PSE-E1A/EIBCN716 CN144 BHG10 PSE-E1A/EIB CN717 CN144 BHG10 PSE-E4 pXC.1 CN135-BHG10ΔEnh/PSE-E1A CN724 BHG10 PSE-E1A, ΔE3 CN725 CN96 CN183 CMV-SV40 T AgPSE-E1A/EIB, CN723 CN144 CN188, CN108, with HR404 BHG10 with ΔE3 CMV-CDPSE-EIA/EIB. CN726 CN144 CN207 ΔE4 (d11006) PSE-E1A/E1B, CN727 CN144CN207 hr404, ΔE4

[0294] Results:

[0295] Virus Construction and Genomic Structure.

[0296] In the initial round of construction three replication competent,prostate-specific adenoviruses were produced. CN706 which contains thePSE driving the expression of the E1A gene, CN711 which contains the PSEdriving the expression of the E1B gene, and CN716 which contains the PSEdriving E1A expression and the PSE driving E1B expression. The viruseswere generated by homologous recombination in 293 cells and cloned twiceby plaque purification. The structure of the genomic DNA was analyzed byPCR and sequencing of the junctions between the inserted sequences andthe Ad genomic sequences. All viruses contained the desired structures.

[0297] Virus Growth in Vitro.

[0298] The growth of the viruses in vitro was characterized by twoassays: a burst size assay to measure the amount of infectious particlesproduced in one round of infection and plaque assays to assess thegrowth of the viruses in various types of cells.

[0299] For the burst size assays either LNCaP cells (a CaP cell linewhich produces PSA) or HBL100 cells (a non-malignant breast epithelialcell line) were infected with virus at a multiplicity of infection (MOI)of 1 (5×105 PFU per sample). At various time points samples wereharvested and the amount of infectious virus present measured by plaqueassays on 293 cells. Table 2 shows that CN706 produced 6.3×10⁶ pfU froman input of 5×10⁵ pfu in LNCaP cells after 48 hours. In HBL100 cells theincrease from the same amount of input virus was to 2.0×10⁶ pfu. CN706then yielded 13 pfu per input infectious particle in LNCaP cells whichwas 3 fold greater than that produced in HBL100 cells over the same timeperiod.

[0300] Burst size assays on CN711 also revealed preferential growth inLNCaP cells versus HBL100 cells (Table 2). In LNCaP cells 5×10⁵ Y pfuinput virus produced 4×10⁷ pfu at 48 hours while in HBL100 cells 8×10⁶pfu were obtained at 48 hours. This represented a 40 fold increase invirus in LNCaP cells or a 5 fold greater yield than in HBL100 cells.

[0301] The differential in virus production for CN716 showed a widerdisparity between the two cell lines. In LNCaP cells 1.7×10⁷ pfu wereobtained after 48 hours while in HBL100 cells 8×10⁵ pfu were obtained atthe same time point. Therefore in LNCaP cells 34 infectious particleswere produced for each input particle at 48 hours while for HBL100 1.6infectious particles was produced.

[0302] These results indicate that the expression of the-early genes E1Aand E1B can be controlled by the inserted PSE. To further characterizethis regulation, production of CN706 virus was assayed by the burstassay in LNCaP cells in the presence or absence of the testosteroneanalog R1881. Since the PSE is highly active in the presence ofandrogens but essentially inactive in the absence of androgens, theproduction of early proteins controlled by the PSE and therefore theproduction of virus should be sensitive to androgen levels. As shown inTable 3 in the absence of R1881, 3×10⁶ pfu were obtained at 48 hours fora three fold increase over input virus. In the presence of 1 nM or 10 nMR1881 two to three fold more pfu were obtained at 48 hours. In contrast,with wild type adenovirus assayed in parallel, no difference was evidentin pfu obtained in the presence or absence of R1881. TABLE 2 BurstAssays LNCaP HBL100 CN706 6.3 × 10₆ 2.0 × 10⁶ CN711   4 × 10₇   8 × 10⁶CN716 1.7 × 10₇   8 × 10⁵

[0303] TABLE 3 R1881 induction 0 nM R1881 1 nM R1881 10 nM R1881 CN706 3× 10₆ 8 × 10₆ 5 × 106

[0304] To further assess the growth selectivity of CN706, CN711, andCN716, the viruses were analyzed in plaque assays in which a singleinfectious viral particle produces a visible plaque by multiple roundsof infection and replication. The results of a representative assay areshown in Table 4. TABLE 4 Plaque assay Cell line 293 LNCaP HBL100 TSUA549 CN702 2.3 × 10₅ 4.1 × 10₅ 4.3 × 10₅ 1.1 × 10₆ 5.1 × 10₅ CN706 2.3 ×10₅ 4.4 × 10₄ 1.7 × 10₃ 5.4 × 10₄ 2.9 × 10₄ CN711 2.3 × 10₅ 5.5 × 10₅2.7 × 10₅ 1.6 × 10₅ 2.6 × 10₅ CN716 2.3 × 10₅ 6.9 × 10₅ 2.7 × 10₃ 4.4 ×10₃ 4.1 × 104

[0305] Virus stocks were diluted to equal pfu/ml, then used to infectmonolayers of cells for 1 hour. The inoculum was then removed and thecells were overlayed with semisolid agar containing medium and incubatedat 37° C. for one week. Plaques in the monolayer were then counted andtiters of infectious virus on the various cells were calculated. Thedata were normalized to the titer of CN702 on 293 cells.

[0306] The wild type virus CN702 showed approximately equal titers oneach of the five cell lines. In contrast, each of the PSE modifiedviruses displayed a variable pattern of growth on the different celltypes. CN706 grew to a 10 fold lower titer on LNCaP cells as on 293cells, however, its titer on HBL100 cells was 260 fold lower than on 293cells. On the non-PSA secreting CaP cell line TSU the titer of CN706 wasapproximately the same as on LNCaP cells which do secrete PSA.Similarly, the titer on the lung cell line A549 was also close to thaton LNCaP cells. The virus CN711 displayed no significant difference intiter on the cell lines tested.

[0307] The data for the CN716 virus revealed a marked selectivity forgrowth in the LNCaP cell line. This virus grew well in LNCaP cells,reaching an even higher titer than on 293 cells. Growth of the virus onother cell lines was significantly lower, 18 fold lower on the nexthighest titer line, A549. The greatest differential was on HBL100 cells,where the titer was 225 fold lower relative to that on LNCaP cells. Thedata from the burst size assay and the plaque assay demonstrate thathuman adenovirus can be modified using the PSE to develop viruses withselective growth properties for PSA secreting CaP cells.

EXAMPLE 2 Treatment of LNCaP Tumor Xenografts

[0308] The ultimate objective in the development of prostate-specificviruses is to treat patients with prostate disease. The feasibility ofthis objective was tested using LNCaP tumor xenografts grownsubcutaneously in Balb/c nu/nu mice. The test viruses were inoculatedinto the mice either by direct intratumoral injection of approximately10⁸ pfu of virus in 0.1 ml PBS+10% glycerol or intravenously via thetail vein. Tumor sizes were measured and, in some experiments, bloodsamples were taken weekly.

[0309] The effect of intratumoral injection of CN706 on tumor size andserum PSA levels was compared to sham treatment. The sizes of the CN706treated tumors continued to increase for two weeks, then progressivelydecreased for the duration of the experiment. At the end of theexperiment all of the CN706 treated tumors (10 total) had diminished insize and five mice were cured of their tumor. In contrast, the buffertreated tumors continued to grow for the duration of the experiment,reaching approximately twice their original size by 42 days.

[0310] Previously published results have shown that serum PSA levelscorrelate with tumor size in the LNCaP tumor xenograft model.Measurement of PSA levels in the mice with tumors treated with CN706indicated a rise in PSA levels one week after treatment, followed by asteady decline in PSA levels out to 35 days. Serum PSA levels increasedduring the course of the experiment, averaging over 250 ng/ml at 35days.

[0311] While it is likely that a therapeutic based on the virusesdescribed here would be given intralesionally, it would also bedesirable to determine if the virus can affect tumor growth followingintravenous administration. If so, then it is conceivable that the viruscould be used to treat metastatic tumor deposits inaccessible to directinjection. Groups of five mice bearing LNCaP tumors were inoculated with10⁸ pfu of CN706 by tail vein injection, or 10⁸ pfu of areplication-defective adenovirus (CMV-LacZ) to control for non-specifictoxic effects of the virus, or with buffer used to carry the virus.Tumors in mice treated with buffer or CMV-LacZ continued to grow for theduration of the experiment, ultimately reaching approximately five timestheir original size on average. Tumors in mice treated with CN706 grewslightly between the time of inoculation and the first measurement at 7days, then the average tumor size diminished to approximately 75% of theoriginal tumor volume by day 42.

[0312] Treatment of LNCaP tumors in nude mice with CN711 resulted in asimilar outcome to treatment with CN706. In the CN711 treated animals (5total) the tumors continued to grow between inoculation and day 8.Thereafter the average tumor size diminished, reaching 65% by day 49.The average tumor size of the buffer treated mice (4 total) increasedthrough the duration of the experiment, reaching 300% of the originaltumor volume by 49 days.

[0313] The same experimental protocol was used to test the CN716 virusin LNCaP tumors. Mice were inoculated with PBS+10% glycerol, CN716, orCN702. The tumors in the buffer mice grew rapidly and the mice weresacrificed due to large tumor sizes after three weeks. Tumors treatedwith CN702 continued to grow for two weeks, then diminished in size to80% of their original volume by day 42. Tumors treated with CN716remained at their original size for one week, then diminished in size to40% of their original size by day 42. At the end of the experiment 2 ofthe 4 mice treated were cured of their tumors.

EXAMPLE 3 Construction of Replication-Competent Adenoviral Vectors inWhich Adenoviral Genes are Under Transcriptional Control ofAlpha-Fetoprotein TRE

[0314] A replication-competent adenoviral vector, CN733, was constructedin which multiple copies of the Alpha Fetoprotein TranscriptionalResponse Element (AFP-TRE) were placed upstream of adenovirus genes E1Aand E1B, as shown schematically in FIG. 1. AFP-TRE is depicted in SEQ IDNO: 44. An alternative AFP-TRE is depicted in SEQ ID NO: 45.

[0315] Cloning Strategy for Vector Construction

[0316] A human embryonic kidney cell line, 293, efficiently expressesE1A and E1B genes of Ad5 and exhibits a high transfection efficiencywith adenovirus DNA. For these experiments, 293 cells wereco-transfected with one left end Ad5 plasmid and one right end Ad5plasmid. Homologous recombination generates adenoviruses with therequired genetic elements for replication in 293 cells which provide E1Aand E1B proteins in trans to complement defects in synthesis of theseproteins.

[0317] The plasmids to be combined were co-transfected into 293 cellsusing cationic liposomes such as Lipofectin (DOTMA:DOPE™, LifeTechnologies) by combining the two plasmids, then mixing the plasmid DNAsolution (10

g of each plasmid in 500

l of minimum essential medium (MEM) without serum or other additives)with a four-fold molar excess of liposomes in 200

1 of the same buffer. The DNA-lipid complexes were then placed on thecells and incubated at 37° C., 5% CO₂ for 16 hours. After incubation themedium was changed to MEM with 10% fetal bovine serum and the cells arefurther incubated at 37° C., 5% CO₂, for 10 days with two changes ofmedium. At the end of this time the cells and medium were transferred totubes, freeze-thawed three times, and the lysate was used to infect 293cells at the proper dilution to detect individual viruses as plaques.

[0318] Plaques obtained were plaque purified twice, and viruses werecharacterized for presence of desired sequences by PCR and occasionallyby DNA sequencing. For further experimentation, the viruses werepurified on a large scale by cesium chloride gradient centrifugation.

[0319] Using the above procedure, three replication competent,hepatocarcinoma cell-specific adenoviruses were produced: CN732, whichcontains an AFP-TRE driving the expression of the E1A gene; CN733, whichcontains two AFP-TREs driving expression of the E1A and E1B genes; andCN734, which contains an AFP-TRE driving E1B expression. The viruseswere generated by homologous recombination in 293 cells and cloned twiceby plaque purification. The structure of the genomic DNA was analyzed byPCR and sequencing of the junctions between the inserted sequences andthe Ad genomic sequences to confirm that the viruses contained thedesired structures. The structure of the viruses was also confirmed bySouthern blot.

[0320] Table 5-lists the combinations of right end and left end Ad5plasmids used to generate recombinant Ad5 with the desired features.TABLE 5 Adenovirus vectors containing AFP-TRE Virus Name Left EndPlasmid Right End Plasmid E1A-AFP CN732 CN219 BHG10 EIA/EIB-AFP CN733CN224 BHG10 E1B-AFP CN734 CN234 BHG10

[0321] Adenoviral Vector Construction

[0322] Plasmid pXC.1 was purchased from Microbix Biosystems Inc.(Toronto). pXC.1 contains Ad5 sequences from (nucleotide) 22 to 5790. Weintroduced an AgeI site 12 bp 5′ to the E1A initiation codon (Ad5 547)by oligo-directed mutagenesis and linked PCR. To achieve this, pXC.1 wasPCR amplified using primers:

[0323] 5′-TCGTCTTCAAGAATTCTCA (15.133A) (SEQ ID NO: 14), containing anEcoRI site, and 5′-TTTCAGTCACCGGTGTCGGA (15.134B) (SEQ ID NO: 15),containing an extra A to introduce an AgeI site. This created a segmentfrom the EcoRI site in the pBR322 backbone to Ad5 560. A second segmentof pXC.1 from Ad 541 to the XbaI site at Ad nucleotide 1339 wasamplified using primers:

[0324] 5′-GCATTCTCTAGACACAGGTG (15.133B) (SEQ ID NO: 16) containing anXbaI site, and 5′-TCCGACACCGGTGACTGAAA (15.134A) (SEQ ID NO: 17),containing an extra T to introduce an AgeI site. A mixture of these twoPCR-amplified DNA segments was mixed and amplified with primers 15.133Aand 15.133B to create a DNA segment from the EcoRI site to the XbaI siteof pXC.1. This DNA segment encompasses the leftmost 1317 bases of Adsequence and contains an AgeI site at Ad 547. This DNA segment was usedto replace the corresponding segment of pXC.1 to create CN95.

[0325] An EagI site was created upstream of the E1B start site byinserting a G residue at Ad5 1682 by oligonucleotide directedmutagenesis as above. To simplify insertion of an AFP-TRE in the EagIsite the endogenous EagI site in CN95 was removed by digestion withEagI, treatment with mung bean nuclease, and re-ligation to constructCN114. The primers:

[0326] 5′-TCGTCTTCAAGAATTCTCA (15.133A) (SEQ ID NO: 14), containing anEcoRI site, and 5′-GCCCACGGCCGCATTATATAC (9.4) (SEQ ID NO: 46),containing an EagI site, and

[0327] 5′-GTATATAATGCGGCCGTGGGC (9.3) (SEQ ID NO: 47) containing anextra G and an EagI site, and 5′-CCAGAAAATCCAGCAGGTACC (24.020) (SEQID-NO:30), containing a KpnI site, were used to amplify the segmentbetween 1682 and the KpnI site at Ad5,2048: Co-amplification of the twosegments with primers 15.133A and 24.020 yielded a fragment with an EagIsite at Ad5 1682 which was used to replace the corresponding EcoRI/KpnIsite in pXC.1 to construct CN124.

[0328] For construction of CN732, human AFP enhancer domains A and B(included in the region −3954 bp to −3335 bp relative to the AFP capsite) were PCR amplified from human genomic DNA (Clontec, Palo Alto,Calif.) using the following primers: 5′ GTGACCGGTGCATTGCTGTGAACTCTGTA 3′(SEQ ID NO:48) (39.055B) 5′ ATAAGTGGCCTGGATAAAGCTGAGTGG 3′ (SEQ IDNO:49) (39.044D)

[0329] The AFP promoter was amplified from −163 to +34 using thefollowing primers: 5′ GTCACCGGTCTTTGTTATTGGCAGTGGT 3′ (SEQ ID NO:50)(39.055J) 5′ ATCCAGGCCACTTATGAGCTCTGTGTCCTT 3′ (SEQ ID NO:51) (29.055M)

[0330] The enhancer and promoter segments were annealed, and a fusionconstruct was generated using overlap PCR with primers 39.055B and39.055J. This minimal enhancer/promoter fragment was digested with PinA1and ligated with CN124 using the engineered AgeI site 5′ of the E1A capsite to produce CN219. The liver specific viral vector CN732 wasgenerated by homologous recombination by cotransfecting 293 cells withCN219 and BHG10.

[0331] CN733 was constructed by using the following two PCR primers toamplify the enhancer/promoter element described above (−3954 to −3335and −174 to +29): 5′ TATCGGCCGGCATTGCTGTGAACTCT 3′ (SEQ ID NO:52)(39.077A) 5′ TTACGGCCGCTTTGTTATTGGCAGTG 3′ (SEQ ID NO:53) (39.0770)

[0332] The PCR product was digested with EagI and ligated into similarlycut CN219. The resulting plasmid, CN244, contains two identical AFPregulatory elements, one each modulating expression of the E1A gene andthe E1B gene. CN733 was generated by homologous recombination in 293cells by cotransfecting CN224 and BHG10.

[0333] To make CN734, the AFP-TRE regulating the expression of the E1Agene was excised from CN224 by the plasmid with PinA1 and religating thevector. The resulting plasmid, CN234, was co-transfected with BHG10 in293 cells to generate CN734.

[0334] Adenovirus Growth In Vitro

[0335] Growth selectivity of CN732, CN733, and CN734 was analyzed inplaque assays in which is single infectious particle produces a visibleplaque by multiple rounds of infection and replication. Virus stockswere diluted to equal pfu/ml, then used to infect monolayers of cellsfor 1 hour. The inoculum was then removed and the cells were overlayedwith semisolid agar containing medium and incubated at 37° C. for 10days (12 days for Table 8). Plaques in the monolayer were the countedand titers of infectious virus on the various cells were calculated. Thedata were normalized to the titer of CN702 (wild type) on 293 cells. Theresults of four representative assays are shown in Tables 6-9. TABLE 6Plaque assay for 733 (E1A/E1B) Cell line Virus Titer Avg. titerTitre/293 702/733 293 733 2.70 × 10⁶ 2.65 × 10⁶ 1 N/A (control) 733 2.60× 10⁶ 702 1.30 × 10⁶ 1.70 × 10⁶ 1 702 2.10 × 10⁶ Hep3B 733 1.01 × 10⁷1.02 × 10⁷ 3.7 .37 (AFP⁺) 733 1.03 × 10⁷ 702 1.00 × 10⁶ 7.02 × 10⁵ 1.36702 5.00 × 10⁵ HepG2 733 9.70 × 10⁶ 1.04 × 10⁷ 3.92 0.292 (AFP⁺) 7331.10 × 10⁷ 702 1.60 × 10⁶ 1.95 × 10⁶ 1.14 702 2.30 × 10⁶ LNCaP 733 4.00× 10³ 3.00 × 10³ 0.0011 290 (AEP⁻) 733 2.00 × 10³ 702 4.00 × 10⁵ 5.05 ×10⁵ 0.32 702 7.00 × 10⁵ HBL100 733 0 0 0 100-1000 (AFP⁻) 733 0 702 1.00× 10² 3.07 × 10² 0.00022 702 6.40 × 10²

[0336] TABLE 7 CN732, CN733, CN734 Plaque Assay Data Cell line Virus AveTiter Titre/293 7XX/702 293 702  1.2 × 10⁶ 1 (control) 732 6.15 × 10⁵ 1733 2.20 × 10⁶ 1 734 2.50 × 10⁵ 1 Huh-7 702 1.10 × 10⁴ 0.01375 732 1.10× 10⁵ 0.1788 13 733 8.50 × 10⁴ 0.0386 3 734 1.90 × 10⁴ 0.076 6 Sk-Hep-1702 9.00 × 10² 0.00113 732 0 0 0 733 0 0 0 734 1.00 × 10³ 0.004 4 HeLa702 2.45 × 10² 0.00030625 732 0 0 0 733 1.5 6.81 × 10⁻⁷ 0.0022 734 2.50× 10³ 0.01 32 MCF-7 702 3.10 × 10³ 0.003875 732 7.5 1.22 × 10⁻⁵ 0.0031733 2.30 × 10¹ 1.05 × 10⁻⁵ 0.0027 734 1.70 × 10³ 0.0068 2 DLD-1 702 1.70× 10³ 0.00213 732 1.40 × 10¹ 2.28 × 10⁻⁵ 0.011 733 1 4.54 × 10⁻⁷ 0.00021734 1.55 × 10³ 0.0062 3

[0337] TABLE 8 CN732, CN733, CN734 Plaquing Efficiency Cell line VirusTiter 293 702 1 × 10⁷ 732 1 × 10⁷ 733 1 × 10⁷ 734 1 × 10⁷ HepG2 702 5 ×10⁶ (AFP⁺) 732 3 × 10⁶ 733 3 × 10⁶ 734 1 × 10⁷ Sk-Hep-1 702 6 × 10⁴(AFP⁻) 732 0 733 0 734 3 × 10⁴ OVCAR-3 702 8 × 10⁵ (AFP⁻) 732 0 733 0734 3 × 10⁴ HBL-100 702 2 × 10⁶ (AFP⁻) 732 0 733 0 734 1 × 10⁴

[0338] TABLE 9 Plaque assay for CN732, CN733, and CN734 Titer (cellline)/ Cell line Virus Ave Titer Titer 293 CN7XX/CN702 293 702 5.0 × 10⁶1 (control) 732 4.8 × 10⁶ 1 733 3.2 × 10⁶ 1 734 3.0 × 10⁸ 1 HepG2 7022.3 × 10⁷ 4.6 — (AFP⁺) 732 3.2 × 10⁷ 6.7 1.5 733 6.0 × 10⁶ 1.9 0.41 7344.2 × 10⁸ 1.4 0.30 DU145 702 2.2 × 10⁶ 0.44 — (AFP⁻) 732 3.0 × 10⁴0.0063 0.0143 733 3.1 × 10³ 0.00097 0.002 734 1.0 × 10⁷ 0.033 0.075HBL-100 702 4.0 × 10⁵ 0.8 — (AFP⁻) 732 0 — 0 733 0 — 0 734 6.0 × 10⁶0.02 0.025 OVCAR-3 702 3.3 × 10⁵ 0.066 — (AFP⁻) 732 0 — 0 733 0 — 0 7343.1 × 10⁵ 0.001 0.015

[0339] The wild type virus CN702 produced plaques on each of the celllines tested. The number of plaques produced by CN702 was used as a baseline against which to compare plaque formation by CN733.

[0340] In 293 cells growth of the viruses should be independent of thealterations to the E1 region due to the trans complementation in thiscell line. As expected, both CN702 and CN733 produced similar numbers ofplaques on 293 cells.

[0341] Regarding the data from Table 6, in the AFP positive cell linesHep3B and HepG2 CN702 produced similar numbers of plaques relative to293 cells. In contrast, CN733 produced approximately four fold moreplaques in the AFP positive cell lines than in 293 cells. The supernormal level of plaque formation by CN733 in the AFP positive linesindicates that the AFP enhancer is active in these cells.

[0342] In the AFP negative cell lines LNCaP and HBL100 growth of bothviruses was curtailed but to different extents. Wild type CN702 virusproduced plaques in LNCaP cells at approximately 30% of the level seenin 293 cells. In HBL-100 cells CN702 formed plaques at 0.02% of thelevel formed in 293 cells. CN733 plaque formation was diminished evenfurther in these AFP negative cell lines relative to CN702. In LNCaPcells CN733 produced plaques at a level 0.1% of that seen in 293 cells.In HBL100 cells CN733 did not produce plaques at all. In comparison toCN702, the growth of CN733 on AFP negative cell lines was reduced by atleast 100 fold. This compares favorably with previous results where theE1B promoter of Ad40 was shown to specify a differential ofapproximately 100 fold between gut and conjunctival epithelial tissues(Bailey et al., 1994) and with deletion mutants of the E1B gene whichwere shown to specify a 100 fold differential in Ad growth between p53+and p53− cells (Bischoff et al., 1996). Lastly, comparison of the titerof an AFP+ cell type to the titer of an AFP− cell type provides a keyindication that the overall replication preference is enhanced due todepressed replication in AFP− cells as well as the replication in AFP+cells.

[0343] Regarding the data from Table 7, several observations can bemade. First, CN732, CN733, and CN734 all plaque as efficiently in Huh-7cells as CN702. In contrast,,the plaquing efficiency for two of theadenoviruses (CN732 and CN733) decreases dramatically in the non-AFPproducing cell lines included in the experiment. In the non AFPproducing hepatocellular carcinoma cell line Sk-Hep-1, CN732 and CN733produced no plaques at the dilutions tested. The results are similar forthese two viruses in HeLa, MCF-7, and DLD-1. CN702's efficiency in DLD-1cells exceeds CN733's by over 4000 fold.

[0344] With respect to the data in Table 8 (in which titers arenormalized to 1×10⁷ in 293 cells), CN732, CN733, and CN734 plaquedsimilarly to wild type (CN702) in HepG2 cells. However, these virusesplaqued poorly compared to CN702 in cell lines that do not express AFP.CN732 and CN733 produced no plaques at the dilutions tested in SK-Hep-1,OVCAR-3 and HBL-100, thus displaying significant titer differential.This corresponds to at least a 10,000 fold difference with wild type inHBL-100 and OVCAR-3 and a 1,000 fold difference in SK-Hep-1. CN734 alsoplaqued less efficiently than CN702 in OVCAR-3 (25 fold) and HBL-100(200 fold) cells.

[0345] The data of Table 9 suggest that CN732, CN733, and CN734 plaqueas efficiently as CN702 in cells that express AFP. However, they do notplaque as efficiently as CN702 in cell lines that do not express AFP.For example, neither CN732 nor CN733 produced any plaques at thedilutions tested in HBL100 cells or OVCAR-3 cells. CN734's plaquingdifferential was not as striking as CN732's or CN733's in the cell linestested. It plaqued 13-fold, 40-fold, and 67-fold less efficiently thanwild type in DU145, HBL100, and OVCAR-3, respectively.

[0346] The plaque assay data demonstrate that human adenovirus can bemodified using an AFP-TRE to develop viruses with selective growthproperties for AFP producing cells, particularly AFP-producing tumorcells such as hepatic carcinoma cells.

[0347] Western Analysis of E1A Expression from CN733

[0348] In the next experiment, we examined the effect of an AFP-TRE onthe accumulation of E1A protein in CN733 infected cells. We reasonedthat if one of the AFP regulatory regions 10 installed in CN733 wasmodulating the E1A gene, the level of E1A protein in infected cellsshould also be affected. A western blot was conducted to test ourhypothesis.

[0349] CN733's E1A accumulation was evaluated in Huh-7, SK-Hep-1 andDLD-1 cells. Monolayers were infected with either CN702 or CN733 at anMOI of ten and the harvested at various time points after infection.Samples were electrophoresed through a 10% acrylimide gel andtransferred by electrophoresis to a nitrocellulose membrane. E1A proteinwas detected by using the ECL Western Detection system (Amersham,Arlington Heights, Ill.) using the suggested protocol. The primaryantibody used was rabbit anti-Ad2 E1A antibody (Santa CruzBiotechnology, Santa Cruz, Calif.). The results are shown in FIG. 2(A).

[0350] E1A accumulated rapidly in CN702 and CN733 infected Huh-7 cells.A high level of E1A was also detected in CN702 infected DId-1 cells.However, little E1A protein was detected in CN733 infected DId-1 cells.This result is intriguing because it suggests that CN733's poor plaquingefficiency in non AFP producing cell lines could be attributed to itsrestricted E1A expression. These data are consistent with the hypothesisthat the AFP-TRE affects CN733's compromised replication innon-permissive cell types.

[0351] The experiment was repeated using Sk-Hep-1 cells as non AFPproducing cells. Data were obtained after 24 hours post-infection. Theresults are shown in FIG. 2(B). The conclusion of this experiment is thesame as the previous experiment: E1A expression is tightly regulated bythe AFP-TRE.

[0352] Growth of CN733

[0353] CN733's growth in AFP- and non-AFP-producing cells was evaluated.Monolayers of Huh-7, Sk-Hep-1, and DId-1 cells were infected at an MOIof ten with either CN702 or CN733. At various times after infection,duplicate samples were harvested, freeze-thawed three times, and titeredon 293 cells to determine the total virus yield. Virus yield curves forCN702 and CN733 are plotted in FIGS. 3(A)-(C).

[0354] CN702 and CN733 grew efficiently in Huh-7 cells. Huh-7 cellsproduced similar amounts of infectious CN702 and CN733. In contrast,CN733's growth was severely restricted in SK-Hep-1 cells. CN702's titerat the conclusion of the experiment is about 1000 times greater thanCN733's titer. The results were similar in DId-1 cells.

[0355] The growth experiment was also performed to compare growth ofCN732, CN733, and CN734 in HepG2 cells. Monolayers of HepG2 cells wereinfected at a multiplicity of infection (MOI) of two and harvested atvarious times after infection. Samples were titered on 293 cells todetermine the final virus yield. The results are shown in FIGS.4(A)-(C). The data demonstrate that the adenovirus containing AFP-TREsgrow efficiently in this cancer cell line. CN732, CN733, and CN734 eachreach a high final titer at 36 hours post infection that is similar tothat of CN702.

[0356] In another experiment, propagation was evaluated in primaryhepatocytes (hNheps) isolated from a donor (32 year old black male)three days before the start of the experiment. Monolayers of cells wereinfected with virus at an MOI of two, harvested at various times afterinfection and titered on 293 monolayers. The results are shown in FIGS.5(A)-(C). The data suggest that CN732 and CN733 grow less efficiently inhNheps than CN702. CN732's growth is delayed by twenty-four hourscompared to CN702's. At thirty-six hours post infection, there is overten fold more infectious CN702 than CN733. CN733's growth is delayed bythirty-six hours. At thirty-six hours post infection, there is nearly1000 times more infections CN702 than CN733. CN734 grows similarly toCN702. The data also suggest that CN733 has the most restrictivephenotype, followed by CN732 and CN734. Taken together, these resultsalso indicate that an AFP-TRE inserted upstream of the E1A gene may bemore effective in restricting host-range than an AFP-TRE engineeredupstream of the E1B region. The presence of two AFP-TREs is even moreeffective.

[0357] In conclusion, the experiments described above indicate that itis possible to restrict an adenoviral vector's host range to AFPproducing cells. As demonstrated by plaque assay and growth assay, theadenovirus vectors containing an AFP-TRE propagate efficiently in HepG2and Huh-7 cells but poorly in non AFP producing cells.

[0358] Testing Cytotoxic Ability of Adenovirus Vector CN733 on HepG2Tumor Xenographs

[0359] An HCC mouse xenograft model was used to evaluate CN733'spotential as a therapeutic adenovirus for liver cancer. The AFPproducing HCC cell line HepG2 was injected subcutaneously on the rightflanks of Balb/c nu/nu mice. After allowing several weeks for the tumorsto take, each was treated with an intratumoral injection of either1.5×10¹¹ particles of CN733 in PBS, glycerol or buffer alone. Elevenmice bearing HepG2 tumors were treated, six with CN733 and five withbuffer. Tumors were measured weekly until the conclusion of theexperiment. Tumor volume was calculated by multiplying the tumor'slength by the square of its width and dividing the product by two. FIG.6(A) is a graph of average tumor volume for each treatment group vs.time.

[0360] In six weeks, HepG2 tumors challenged with buffer grew to overfive times their original size. In contrast, tumor growth in CN733treated mice was attenuated. One tumor even regressed to 3% of itsmaximum volume. These data suggest that CN733 invaded the tumors anddelivered cytotoxicity.

[0361] In addition to monitoring tumor growth, we harvested serumsamples and assayed AFP levels. The results are shown in FIG. 7. Thedata suggest that serum AFP levels rises more slowly in mice receivingCN733 than in control mice receiving buffer.

[0362] In another experiment, antitumor activity of differentadministrative regimens was compared for CN733. Animals were treatedwith a single intramuoral administration of either buffer (n=8;volume=919 mm³) or 1.5×10¹¹ particles of CN733 (n=8, volume=944 mm³). Athird group of animals was treated with five consecutive daily doses of1.5×10¹¹ particles of CN733 (n=8, volume=867 mm³). Despite the largesystemic virus burden, the mice displayed no obvious signs of toxicity.Tumors were measured weekly by external caliper for four weeks afterinjection. Animals from groups treated with a single dose of CN733 andbuffer were sacrificed four weeks after treatment because of excessivetumor burden. All animals from the group treated with five doses ofCN733 survived until the conclusion of the study. Despite the largesystemic virus burden, these animals showed no obvious signs oftreatment related toxicity. The results are shown in FIG. 6(B). Onaverage, buffer treated tumors increased to three times their initialvolume by four weeks after treatment. Tumors treated with a single doseof CN733 increased to nearly four times their initial volume. Incontrast, tumors treated with five doses of CN733 remained the samevolume. Five out of eight tumors (63%) responded to treatment. Oneanimal had no palpable tumor at the end of the study.

[0363] Statistical analysis using the Students T-test suggests thatthere was no significant difference at any time point between buffertreated animals and those treated with one dose of CN733 (p>0.5).However, there was a significant difference between buffer treatedanimals and those treated with five doses of CN733 beginning at twoweeks post injection (p=0.045) and continuing through four weeks(p=0.034).

[0364] The data suggest that CN733 exhibits significant antitumoractivity in HepG2 nude mouse xenografts. CN733 administered daily forfive consecutive days at a dose of 1.5×10¹¹ particles can cause tumorregression in some animals. A single dose, however, is not sufficient tocause tumor killing.

[0365] In the first experiment, the tumors responded to a single dose ofCN733 but did not appear to respond in the second. The inventors notethat there is often a variation in tumor phenotype (including growthcharacteristics and AFP expression) from experiment to experiment.

[0366] In conclusion, the in vivo experiments suggest that CN733 causessignificant tumor killing in large hepatoma xenografts. Five doses ofintratumorally administered virus induced regression in four out ofeight animals and cured one animal twenty-eight days after injection. Onaverage, buffer treated tumors tripled while CN733 treated tumorsremained the same.

EXAMPLE 4 Construction of Replication-Competent Adenoviral Vectors inWhich Adenoviral Genes are Under Transcriptional Control ofCarcinoembrvonic Antigen (CEA) TRE

[0367] Using the procedure described above in Example 3, threereplication competent, CEA cell-specific adenoviruses were produced:CN741, which contains an CEA-TRE driving the expression of the E1A gene;CN742, which contains two CEA-TREs driving expression, of the E1A andE1B genes; and CN743, which contains an CEA-TRE driving E1B expression.These constructs are shown schematically in FIG. 8. The viruses weregenerated by homologous recombination in 293 cells and cloned twice byplaque purification. The structure of the genomic DNA was analyzed byPCR and sequencing of the junctions between the inserted sequences andthe Ad genomic sequences to confirm that the viruses contained thedesired structures.

[0368] Table 10 lists the combinations of right end and left end Ad5plasmids used to generate recombinant Ad5 with the desired features.TABLE 10 Adenovirus vectors containing CEA-TRE Virus Name Left EndPlasmid Right End Plasmid E1A-CEA CN741 CN266 BHG11 EIA/EIB-CEA CN742CN285 BHG11 EIB-CEA CN743 CN290 BHG11

[0369] A replication-competent adenoviral vector, CN742, was constructedin which copies of the Carcinoembryonic Antigen Transcriptional ResponseElement (CEA-TRE) were placed upstream of adenoviral genes E1A and E1B.

[0370] The Carcinoembryonic Antigen Transcriptional Response Element(CEA-TRE)

[0371] The transcriptional response element of the carcinoembryonicantigen (CEA-TRE), about −402 to about +69 bp relative to thetranscriptional start (SEQ ID NO: 54), was amplified by polymerase chainreaction (PCR) from human genomic DNA using primers:

[0372] 5′ ATT ACC GGT AGC CAC CAC CCA GTG AG 3′ (39.174B, upper primer)(SEQ ID NO: 55) and 5′ TAG ACC GGT GCT TGA GTT CCA GGA AC 3′ (39.174D)(SEQ ID NO: 56).

[0373] A unique restriction site, AgeI, was introduced by the primerpair at the ends of the PCR-amplified product.

[0374] The CEA-TRE PCR fragment was ligated into pGEM-T vector (Promega)which had been linearized with EcoRV. The ligation mixture wastransformed into E. coli DH5α cells. The desired clone, carrying aCEA-TRE fragment, was obtained and designated CN265.

[0375] Construction of CEA-TRE Adenoviruses Comprising One or TwoAdenovirus Genes Under Transcriptional Control of CEA-TRE

[0376] Three replication-competent, CEA cell-specific adenoviruses wereproduced:

[0377] CN741, which contains a CEA-TRE driving the expression of the E1Agene;

[0378] CN742, which contains two CEA-TREs driving expression of both theE1A and E1B genes; and

[0379] CN743, which contains a CEA-TRE driving E1B expression.

[0380] The viruses were generated by homologous recombination in 293cells and cloned by plaque purification. The structure of the genomicDNA was analyzed by PCR and sequencing of the junctions between theinserted sequences and the Ad genomic sequences to confirm that theviruses contained the desired structures.

[0381] CEA-TRE-Driven E1A Adenovirus Plasmid (CN741)

[0382] Briefly, a CEA-TRE fragment was inserted into CN124 (a left-handadenovirus plasmid, described below) to generate CN266, which comprisesthe left-hand end of adenovirus with a CEA-TRE controlling expressing ofthe adenovirus E1A gene. CN266 was recombined with a plasmid carryingthe right-hand portion of adenovirus to generate CN741, which is afull-length adenovirus in which CEA-TRE controls expression ofadenovirus gene E1A.

[0383] In more detail, the CEA-TRE sequence was excised from CN265(described in Example 1) by digestion with PinA1.

[0384] CN124 is a derivative of construct pXC.1, which contains thewild-type (wt) left-hand end of Ad5, from nt (nucleotide) 22 to 5790,including both E1A and E1B [McKinnon (1982) Gene 19:33-42]. PlasmidpXC.1 was purchased from Microbix Biosystems Inc. (Toronto). An AgeIsite was introduced 12 bp 5′ to the E1A initiation codon (Ad5 nt 547) byoligo-directed mutagenesis and linked PCR. To achieve this, pXC.1 wasPCR-amplified using primers:

[0385] 15.133A, 5′-TCGTCTTCAAGAATTCTCA (SEQ ID NO: 14), containing anEcoRI site, and

[0386] 15.134B, 5′-TTTCAGTCACCGGTGTCGGA (SEQ ID NO: 15), containing anextra A to introduce an AgeI site. This created a segment-from the EcoRIsite in the pBR322 backbone to Ad5 nt 560. A second segment of pXC.1from Ad nt 541 to the XbaI site at Ad nucleotide 1339 was amplifiedusing primers:

[0387] 15.133B, 5′-GCATTCTCTAGACACAGGTG (SEQ ID NO: 16) containing anXbaI site, and

[0388] 15.134A, 5′-TCCGACACCGGTGACTGAAA (SEQ ID NO: 17), containing anextra T to introduce an AgeI site.

[0389] These two PCR-amplified DNA segments were mixed and amplifiedwith primers 15.133A and 15.133B to create a DNA segment from the EcoRIsite to the XbaI site of pXC.1. This DNA segment encompasses theleftmost 1317 bases of Ad sequence and contains an AgeI site at Ad nt547. This DNA segment was used to replace the corresponding segment ofpXC.1 to create CN95.

[0390] An EagI site was created upstream of the E1B start site byinserting a G residue at Ad5 nt 1682 by oligonucleotide directedmutagenesis as above. To simplify insertion of a CEA-TRE in the EagIsite, the endogenous EagI site in CN95 was removed by digestion withEagI, treatment with mung bean nuclease, and re-ligation to constructCN114. The following primers were used to amplify the segment between1682 and the KpnI site at Ad5 nt 2048:

[0391]15.133A, 5′-TCGTCTTCAAGAATTCTCA (SEQ ID NO: 14), containing anEcoRI site, and

[0392] 9.4, 5′-GCCCACGGCCGCATTATATAC (SEQ ID NO: 46), containing an EagIsite

[0393] 9.3, 5′-GTATATAATGCGGCCGTGGGC (SEQ ID NO: 47), containing anextra G as well as an EagI site, and 24.020, 5′-CCAGAAAATCCAGCAGGTACC(SEQ ID NO: 30), containing a KpnI site. Co-amplification of the twosegments with primers 15.133A and 24.020 yielded a fragment with an EagIsite at Ad5 nt 1682, which was used to replace the correspondingEcoRI/KpnI site in pXC.1 to construct CN124.

[0394] A CEA-TRE fragment excised from CN265 (see above) by digestionwith PinA1 was ligated into similarly digested CN124 (which contains theleft hand end of the adenovirus) to generate CN266. CN266 is a vectorcomprising the left-hand portion of adenovirus, in which a CEA-TRE isinserted upstream of and controls expression of E1A.

[0395] The full-length CEA-E1A virus, designated CN741, was constructedby homologous recombination of CN266 and BHG11, which contains the righthand side of Adenovirus 5. Briefly, the plasmid CN266 was digested withPvul; BHG11, with Clal. Equivalent amounts (5

g) of each linearly cut plasmid were transfected into 293 cells with a4-fold excess of cationic liposomes such as Lipofectin DOTAP/DOPE (1:1).293 is a human embryonic kidney cell line which efficiently expressesthe E1A and E1B genes of Ad5 and exhibits a high transfection efficiencywith adenovirus DNA. 8 days after infection, viral plaques were observedon the cell monolayer; cells/viruses were harvested, freeze-thawed3×,centrifuged to pellet the cellular debris, and the supernatantcollected. CN741, the full-length adenovirus in which a CEA-TRE controlsE1A expression, was plaque-purified three times.

[0396] In an alternative protocol for transfection of right- andleft-hand adenovirus plasmids into 293 cells, the plasmids are firstcombined, then the plasmid DNA solution (10 μg of each plasmid in 200

l of minimum essential medium without serum or other additives) is mixedwith an 4-molar excess of liposomes (e.g., DOTAP/DOPE) in 200

1 of the same buffer. The DNA-lipid complexes are then placed on thecells and incubated at 37° C., 5% CO₂ for 16 hours. After incubation,the medium is changed to MEM with 10% fetal bovine serum and the cellsare further incubated at 37° C., 5% CO₂, for two weeks with two changesof medium. At the end of this time the cells and medium are transferredto tubes, freeze-thawed three times, and the lysate is used to infect293 cells at the proper dilution to detect individual viruses asplaques. Plaques obtained were plaque-purified twice, and viruses werecharacterized for presence of desired sequences by PCR and occasionallyby DNA sequencing. For further experimentation the viruses are preparedon a larger scale by cesium chloride gradient centrifugation.

[0397] Several clones of CN741, the full-length adenovirus in which aCEA-TRE controls E1A expression, were characterized by PCR, SouthernBlot, and the plaque assay for specificity.

[0398] 1. PCR: Primers were used to amplify the region of clones ofCN741 starting upstream of the CEA insert in the E1A region (primer39.141C: 5′ ATT TGT CTA GGG CCG GGA CTT 3′ (SEQ ID NO: 57)) anddownstream at the 3′ end of the E1B region (primer 39.141H : 5′ CGC GCGCAA AAC CCC TAA ATA AAG 3′ (SEQ ID NO: 58)) of adenovirus. The amplifiedfragment is 4249 bp. The following clones tested positive by PCR:46.130.7.4., 46.130.8.3, 46.130.9.1.1, 46.130.9.2.1, 46.130.9.3.1, and46.130.9.4.1.

[0399] 2. Southern blot: Positive clones of CN741 were furthercharacterized by Southern blot. Viral DNA of CN741 clones was digestedby the following enzymes: ScaI, AflII, and AflII/XbaI. The viral DNA wasprobed with a randomly primed fragment of E1A. The correct fragmentswere as follows: ScaI digest, 926 and 5645 bp; AflII digest, 4011 bp;and AflII/XbaI digest, 1817 bp. Each positive clone displayed thecorrect fragment pattern.

[0400] 3. Plaque assay: The plaque assay is described in Example 2.

[0401] These assays confirmed the identity of CN741, the full-lengthadenovirus in which a CEA-TRE controls E1A expression.

[0402] CEA-TRE-Driven E1B Adenovirus Plasmid (CN743)

[0403] Briefly, a CEA-TRE fragment was inserted into CN124 (a left-handAd vector, described above) to generate CN290, which comprises theleft-hand end-of adenovirus with a CEA-TRE controlling expressing of theadenovirus E1B gene. CN290 was recombined with a plasmid carrying theright-hand portion of adenovirus to generate CN743, which is afull-length adenovirus in which CEA-TRE controls expression ofadenovirus gene E1B.

[0404] In more detail, the CEA-TRE was obtained as an EagI fragment fromCN284 (described below). This fragment was isolated by gelelectrophoresis and inserted into CN124, similarly cut with EagI. CN124,also described above, contains the left-hand portion of Adenovirus 5,with an artificial EagI site upstream of the E1B start site. Theresulting clone, designated CN290, has a CEA-TRE inserted upstream ofthe E1B in a left-hand portion of adenovirus. The identity of CN290 wasconfirmed by restriction digest (ScaI: 2937 and 7406 bp; SmaI: 180, 783,2628, and 6752 bp).

[0405] CN743 was generated by homologous recombination byco-transfecting 293 cells, which produces E1B, with CN290 and BHG11,which contains the wt right hand portion of Ad5. Thus, CN743 is afull-length adenoviral genome in which gene E1B is under control of aCEA-TRE.

[0406] Construction of Adenovirus Vectors in Which Expression of TwoAdenovirus Genes Are Each Controlled by a CEA-TRE (CN742)

[0407] Briefly, a CEA-TRE fragment was inserted upstream of the E1B genein construct CN266, which already had a CEA-TRE fragment insertedupstream of E1A. The resulting plasmid was designated CN285 andcontained a left-hand portion of adenovirus with separate copies of aCEA-TRE driving expression of E1A and E1B. CN285 was recombined with aright-hand portion of adenovirus to generate CN742, which is afull-length adenovirus in which expression of both E1A and E1B iscontrolled by CEA-TRE.

[0408] In more detail, CN285 was constructed by amplifying the CEA-TREinserted into the E1A region (e.g., CN266) by PCR using primers:

[0409] 5′ TM CGG CCG AGC CAC CAC CCA 3′ (39.180A, upper primer) (SEQ IDNO: 59) and

[0410] 5′ TAT CGG CCG GCT TGA GTT CCA GG 3′ (39.180B, lower primer) (SEQID NO: 60). The unique restriction site EagI was introduced by theprimer pair at the ends of the PCR-amplified product. The PCR productwas ligated into pGEM-T Vector (Promega), and the resultant plasmiddesignated CN284.

[0411] The EagI CEA-TRE fragment was excised from CN284 and isolated bygel electrophoresis. The CEA-TRE fragment was ligated into CN266 whichhad been cut with EagI. CN266 (described above) is a left-hand portionof adenovirus in which a CEA-TRE controls expression of E1A. Theresulting clone was confirmed by restriction digest (ScaI: 1682, 1732,and 7406 bp; SmaI: 783, 899 2628, and 6330 bp). The clone was designatedCN285, which represents a left-hand portion of adenovirus in which bothE1A and E1B are under control of separate CEA-TREs.

[0412] CN742 was generated by homologous recombination byco-transfecting 293 cells with CN285 and BHG11, which has the wt righthand portion of adenovirus. Thus, construct CN742 is a full-lengthadenoviral genome with genes E1A and E1B both under control of aCEA-TRE.

[0413] In short, full-length adenoviruses were constructed in which oneor two adenoviral early genes were under transcriptional control of aCEA-TRE.

[0414] Comparative Testing of Virus Growth in Vitro

[0415] Growth selectivity of CN741, CN742 and/or CN743 (full-lengthadenoviruses in which one or two early genes is under control of aCEA-TRE) is analyzed in plaque assays in which a single infectiousparticle produces a visible plaque by multiple rounds of infection andreplication. Virus stocks are diluted to equal pfu/ml, then used toinfect monolayers of cells for 1 hour. Comparison of. normalized titresin cells that allow a CEA-TRE to function and cells that do not allow aCEA-TRE to function indicates replication preference. Cells chosen forthis study are cells that allow a CEA-TRE to function, such as NCIH508,LoVo, SW1463, MKN1, MKN28, MKN45 and cells that do not allow suchfunction, such as HuH7 or HeLa. The inoculum is then removed and thecells are overlayed with semisolid agar containing medium and incubatedat 37° C. for one week. Plaques in the monolayer are then counted andtiters of infectious virus on the various cells are calculated. The dataare normalized to the titer of CN702 (wild type) on 293 cells.

[0416] Full-length adenovirus CN741, in which transcription of E1A isunder control of CEA-TRE, was tested in this way. Clone 46.130.8.3 wasused, and CN702 (wt adenovirus) was a control. Plaques observed on celllines were normalized to infectivity on control 293 Cells. The ratio ofnormalized plaques of CN741 and CN702 were compared to evaluate plaquepreference in cell types. Table 2 depicts the plaque assay results.Cells examined were 293 (CEA-deficient), LoVo (CEA-producing), OVCAR(CEA-deficient), HBL100 (CEA-deficient), and HepG2 (CEA-producing). Wehave found that OVCAR and HBL100 cells do not express levels of CEAdetectable by ELISA, using a standard protocol with a kit purchased fromGenzyme. However, while we also found that HepG2 cell do not produce CEAdetectable in the ELISA test, Zhai et al. [(1990) Gastroenter. 98:470-7]showed that HepG2 cells do produce CEA, as detectable by the PAP andavidin-biotin technique. TABLE 11 Plaque assay results of CN741(CEA-E1A) on human cell lines Normalized Plaques Normalized PlaquesRatio of Cell Line CN702 (wt) CN741 (CEA-E1A) CN741/CN702 293 1.0 1.01.0 LoVo 1.5 0.579 0.39 OVCAR 1.2 0.372 0.31 HBL100 0.75 0.085 0.11HepG2 1.75 0.69 0.39

[0417] The plaque assay results in Table 11 indicate that the growthpattern of CN741 has been altered by the introduction of a CEA-TRE. Ineach cell line, the growth of the CN741 virus is reduced in comparisonto wild-type adenovirus CN702. The ratio of CN741/CN702 in theCEA-proficient cell lines LoVo and HepG2 were similar. Importantly,there was a 4-fold reduction in the ability of CN741 to replicate in theCEA-deficient cell line HBL100 cells. These data seem to indicate thatCN741 has a greater ability (i.e., more specificity for replication) inCEA-proficient cells (LoVo and HepG2) than in CEA-deficient cells(HBL100).

[0418] Curiously, the CN741/CN702 ratio was similar in OVCAR(CEA-deficient) to that in CEA-producing cells. This suggests thatreplication of the CEA-E1A adenovirus relative to wt virus in OVCAR(CEA-deficient) was similar to that in CEA-producing cells. There areseveral possible explanations for this finding. Note that HepG2, asstated above, was determined to be CEA-deficient a CEA ELISA assay, butrevealed to be CEA-proficient by the PAP and avidin-biotin technique.The ELISA method may be similarly insufficient to detect low levels ofCEA present in OVCAR. Alternatively, it is possible that OVCAR cellsalso produce CEA, but the protein is expressed too transiently or tooquickly degraded to be detectable by ELISA, yet is somehow able to allowactivation of transcription of a CEA-TRE and replication of CN741.

EXAMPLE 5 Construction of Replication-Competent Adenoviral Vectors inWhich Adenoviral Genes are Under Transcriptional Control of Mucin TRE

[0419] Adenoviral vectors in which a MUC1-TRE controls expression of E1Aand/or E1B were constructed. A construct (CN226) was built in whichMUC1-TRE controls E1A. In another vector, a MUC1-TRE was insertedupstream of the E1B gene in CN226. A third vector CN237 was constructed,in which MUC1-TRE mediates E1A and E1B expression.

[0420] Construction of CN226 (MUC1-TRE E1A)

[0421] Briefly, CN226, in which MUC1-TRE controls E1A expression, wasconstructed as follows. The MUC1-TRE region of SEQ ID NO: 61 wasamplified from human genomic DNA (Clonetech) by PCR (Perkin Elmer 2400)with the following primer pairs: 5′ TAA TCC GGA CGG TGA CCA CTA GAG GG3′ (39.088A, upper primer-SEQ ID NO: 62) and 5′ TAT TCC GGA TCA CTT AGGCAG CGC TG 3′ (39.088B, lower primer-SEQ ID NO: 63). The primers wereconstructed with BspEI ends, which are compatible with the AgeI site inCN124. CN124 is a derivative of construct pXC.1, which contains thewild-type left-hand portion of Adenovirus 5 (Ad5), from nt 22 to 5790,including both E1A and E1B (McKinnon (1982) Gene 19:33-42). CN124 alsohas, among other alterations, an artificial AgeI site at Ad5 nt 547(just upstream of the E1A transcriptional start at nt 498 and the E1Acoding segment beginning with ATG at 610). CN124 also contains anartificial EagI site at Ad5 nt 1682, or just upstream of the E1B codingsegment. To construct CN124 from pXC.1, we introduced an AgeI site 12 bp5′ to the E1A initiation codon (Ad5 547) by oligonucleotide-directedmutagenesis and linked PCR. To achieve this, pXC.1 was PCR amplifiedusing primers:

[0422] 5′-TCGTCTTCAAGAATTCTCA (15.133A) (SEQ ID NO: 14), containing anEcoRI site, and

[0423] 5′-TTTCAGTCACCGGTGTCGGA (15.134B) (SEQ ID NO: 15), containing anextra A to introduce an AgeI site. This created a segment from the EcoRIsite in the pBR322 backbone to Ad5 560. A second segment of pXC.1 fromAd 541 to the XbaI site at Ad nucleotide 1339 was amplified usingprimers: 5′-GCATTCTCTAGACACAGGTG (15.133B) (SEQ ID NO: 16) containing anXbaI site, and 5′-TCCGACACCGGTGACTGAAA (15.134A).(SEQ ID-NO: 17),containing an extra T to introduce an AgeI site. A mixture of these twoPCR-amplified DNA segments was mixed and amplified with primers 15.133Aand 15.133B to create a DNA segment from the EcoRI site to the XbaI siteof pXC.1. This DNA segment encompasses the leftmost 1317 bases of Adsequence and contains an AgeI site at Ad 547. This DNA segment was usedto replace the corresponding segment of pXC.1 to create CN95.

[0424] An EagI site was created upstream of the E1B start site byinserting a G residue at Ad5 1682 by oligonucleotide directedmutagenesis as above. To simplify insertion of an AFP-TRE in the EagIsite the endogenous EagI site in CN95 was removed by digestion withEagI, treatment with mung bean nuclease, and re-ligation to constructCN114. The primers: 5′-TCGTCTTCAAGAATTCTCA (15.133A) (SEQ ID NO: 14),containing an EcoRI site, and 5′-GCCCACGGCCGCATTATATAC (9.4) (SEQ ID NO:46), containing an EagI site, and 5′-GTATATAATGCGGCCGTGGGC (9.3) (SEQ IDNO: 47) containing an extra G and an EagI site, and5′-CCAGAAAATCCAGCAGGTACC (24.020) (SEQ ID NO: 30), containing a KpnIsite, were used to amplify the segment between 1682 and the KpnI site atAd5 2048. Co-amplification of the two segments with primers 15.133A and24.020 yielded a fragment with an EagI site at Ad5 1682 which was usedto replace the corresponding EcoRI/KpnI site in pXC.1 to constructCN124.

[0425] Amplification of the MUC1-TRE utilized an annealing temperatureof 55° C. (30 cycles) with an extension temperature and time of 72° C.for 60 seconds. PCR products were purified with the QIAQuick Spin PCRPurification Kit (Qiagen). The MUC1-TRE PCR product was digested withBspI and ligated in front of the E1A region of CN124, which had beenlinearized with PinA1 to AgeI ends. Ligation of the insert into thevector destroyed the AgeI restriction site. The resulting clone wasconfirmed by restriction digest : HindIII/PinA1, 1278, 1524, 6730 bp.The MUC1-E1A adenoviral clone was designated CN226 (ref. 39.112).

[0426] MUC1 E1B Ad5 Plasmid (CN292)

[0427] An adenovirus vector in which the expression of the E1B gene isunder control of the MUC1-TRE was constructed as follows.

[0428] The plasmid CN237 (MUC1 E1A/E1B, described above) was digestedwith EagI to excise the MUC1-TRE EagI fragment. The MUC1-TRE fragmentwas isolated by gel electrophoresis (1.2 % SeaKem Agarose) onto DEAEfilter paper and ligated into CN124 (described above) which had beenlinearized with EagI. The resulting clone was confirmed by restrictiondigest: PinA1, 1924 and 7826-bp; HindIII/PinA1 807, 1199, 1924, 6730 bp.The MUC1 E1B clone has been designated CN292 (ref. 46.050).

[0429] Construction of MUC1-TRE E1A/E1B construct CN237

[0430] To construct adenoviral vector CN237, in which a MUC1-TREcontrols expression of both E1A and E1B, a second MUC1-TRE was insertedupstream of the E1B gene in construct CN226, which already contained aMUC1-TRE controlling expression of E1A.

[0431] In more detail, a fragment containing the MUC1-TRE with EagI endswas obtained by PCR of CN226 with the following primer pairs: 5′ TM CGGCCG CGG TGA CCA CTA GAG 3′ (39.120A, upper primer-SEQ ID NO: 64) and 5′TAT CGG CCG GCA GM CAG ATT CAG 3′ (39.120B, lower primer-SEQ ID NO: 65).Amplification of the MUC1-TRE containing EagI ends utilized an annealingtemperature of 55° C. (30 cycles) with an extension temperature and timeof 72° C. for 60 seconds. PCR products were purified with the QIAQuickSpin PCR Purification Kit (Qiagen). The MUC1-TRE PCR product wasdigested with EagI and ligated in front of the E1B region of CN226,which had been linearized with EagI, which cuts just upstream of the E1Bcoding segment. The resulting clone was confirmed by restriction digest:PinA1, 1997 and 9453 bp; SmaI, 179, 980, 1917, 2711, 6562 bp. The MUC1E1A/E1B clone has been designated CN237 (ref. 39.143).

[0432] Homologous Recombination of CN226, CN237, and CN292 with BHG10 orBHG11

[0433] Adenovirus containing the MUC1-TRE regulating expression of E1A,E1B, and E1A/E1B, in the context of the otherwise intact genome havebeen obtained through homologous recombination with the right hand endplasmids BHG10 and/or BHG11 [Bett. et al. (1994); Microbix BiosystemsInc., Toronto] in 293 cells (human embryonic kidney cell line). Theplasmids (e.g. CN226 and BHG10; or CN237 and BHG10, etc.) have beenco-transfected into 293 cells via cationic lipids (DOTAP:DOPE™ 1/1 moleratio) by a standard transfection protocol, including, but not limitedto, that detailed below.

[0434] Adenoviral vector CN735, in which E1A is under control ofMUC1-TRE, was obtained by homologous recombination of CN226 and BHG10.An adenoviral vector, to be designated CN744, comprising E1B undercontrol of MUC1-TRE, can be obtained by homologous recombination ofCN292 and BHG11. Adenoviral vector CN736, in which both E1A and E1B areunder control of MUC1-TRE, was obtained by homologous recombination ofCN237 and BHG10. These plasmids and adenoviral vectors are diagrammed inFIG. 9.

EXAMPLE 6 Construction of Replication-Competent Adenoviral Vectors inWhich Adenoviral Genes are Under Transcriptional Control of Probasin TRE

[0435] Adenoviral vectors in which a PB-TRE was placed upstream of E1Aand/or E1B were constructed.

[0436] The Probasin Transcriptional Response Element (PB-TRE)

[0437] The 454 nucleotide fragment (nt about −426 to about +28) of therat PB-TRE, which contains two androgen response elements (ARE sites), aCAAT box and a TATAA box (FIG. 10, SEQ ID NO: 9), was amplified bypolymerase chain reaction (PCR) using rat genomic DNA as template andthe synthetic oligonucleotides:

[0438] 42.2.1 (SEQ ID NO: 66):

[0439] 5′-GATCACCGGTAAGCTTCCACAAGTGCATTTAGCC-3′,

[0440] PinA1 site underlined,

[0441] and

[0442] 42.2.2 (SEQ ID NO: 67):

[0443] 5′-GATCACCGGTCTGTAGGTATCTGGACCTCACTG-3′ or oligonucleotides

[0444] 42.2.3 (SEQ ID NO: 68):

[0445] 5′-GATCCGGCCGAAGCTTCCACAAGTGCATTTAGCC-3′,

[0446] EagI site underlined,

[0447] and

[0448] 42.2.4 (SEQ ID NO: 69):

[0449] 5′-GATCCGGCCGCTGTAGGTATCTGGACCTCACTG-3′.

[0450] The oligonucleotides created a unique PinA1 (AgeI) site (A/CCGGT)or EagI site (C/GGCCG) at both ends of the PCR fragments. The PCRfragments were ligated into the pGEM-T vector (Promega) to generateplasmids CN249 and CN250. Similarly, CN256 was created using the samestrategy but the PB-TRE fragment was ligated into the pCRT vector(Invitrogen); CN271 is identical to CN250 but with a HindIII site at the5′-end. These plasmids provide the PB-TRE DNA fragments for theconstructs reported below. In some of the adenovirus vectors describedbelow, the endogenous (adenoviral) TREs were not deleted; rather, ineach construct, the PB-TRE was inserted between the endogenous TRE(e.g., the E1A TRE) and its respective coding segment (e.g., the E1Acoding segment). In other vectors, the endogenous (Ad5)promoter-enhancer has been deleted, and the prostate-specificpromoter-enhancer placed immediately upstream of an early gene.

[0451] PB-TRE-driven E1A Ad5 plasmid (CN251)

[0452] An adenovirus vector in which expression of an early gene, E1A,is under control of PB-TRE was constructed as follows.

[0453] CN124 is a derivative of construct pXC.1, which contains thewild-type left-hand end of Ad5, from nt 22 to 5790, including both E1Aand E1B (McKinnon (1982) Gene 19:33-42). CN124 also has, among otheralterations, an artificial PinA1 site at Ad5 nt 547 (between the E1Atranscriptional start at nt 498 and the E1A coding segment beginningwith ATG at 560).

[0454] To construct CN124 from pXC.1, we introduced an AgeI site 12 bp5′ to the E1A initiation codon (Ad5 547) by oligonucleotide-directedmutagenesis and linked PCR. To achieve this, pXC.1 was PCR-amplifiedusing primers:

[0455] 5′-TCGTCTTCAAGAATTCTCA (15.133A) (SEQ ID NO: 14), containing anEcoRI site,

[0456] and

[0457] 5′-TTTCAGTCACCGGTGTCGGA (15.134B) (SEQ ID NO: 15), containing anextra A to introduce an AgeI site. This created a segment from the EcoRIsite in the pBR322 backbone to Ad5 560. A second segment of pXC.1 fromAd 541 to the XbaI site at Ad nucleotide 1339 was amplified usingprimers:

[0458] 5′-GCATTCTCTAGACACAGGTG (15.133B) (SEQ ID NO: 16) containing anXbaI site,

[0459] and

[0460] 5′-TCCGACACCGGTGACTGAAA (15.134A) (SEQ ID NO: 17), containing anextra T to introduce an AgeI site. These two PCR-amplified DNA segmentswere mixed and amplified with primers 15.133A and 15.133B to create aDNA segment from the EcoRI site to the XbaI site of pXC.1. This DNAsegment encompasses the leftmost 1317 bases of Ad sequence and containsan AgeI site at Ad 547. This DNA segment was used to replace thecorresponding segment of pXC.1 to create CN95.

[0461] An EagI site was created upstream of the El B start site byinserting a G residue at Ad5 1682 by oligonucleotide directedmutagenesis as above. To simplify insertion of an PB-TRE in the EagIsite, the endogenous EagI site in CN95 was removed by digestion withEagI, treatment with mung bean nuclease, and re-ligation to constructCN1 14. The primers:

[0462] 5′-TCGTCTTCAAGMTTCTCA (15.133A) (SEQ ID NO: 14), containing anEcoRI site,

[0463] and

[0464] 5′-GCCCACGGCCGCATTATATAC (9.4) (SEQ ID NO: 46), containing anEagI site, and

[0465] 5′-GTATATMTGCGGCCGTGGGC (9.3) (SEQ ID NO: 47) containing an extraG and an EagI site, and

[0466] 5′-CCAGAAAATCCAGCAGGTACC (24.020) (SEQ ID NO: 30), containing aKpnI site, were used to amplify the segment between 1682 and the KpnIsite at Ad5 2048. -Co-amplification of the two segments withprimers-15.133A and 24.020 yielded a fragment with an EagI site at Ad51682 which was used to replace the corresponding EcoRI/KpnI site inpXC.1 to construct CN124.

[0467] CN124 was linearized with PinA1 and dephosphorylated with calfintestinal alkaline phosphatase (New England Biolabs). CN249 wasdigested with PinA1 to free the PB-TRE fragment. The PB-TRE fragment wasthen ligated into the PinA1-linearized CN124, producing CN251. CN253 issimilar to CN251 except for the PB-TRE fragment is in the reverseorientation.

[0468] Thus, construct CN251 contains the PB-TRE inserted upstream ofand operably linked to the E1A coding segment in the Adenovirus 5genome.

[0469] PB-TRE-driven E1B Ad5 plasmid (CN254)

[0470] An adenovirus derivative in which the expression of the E1B geneis under control of the PB-TRE was constructed as follows.

[0471] CN124, described above, also contains an artificial EagI site atAd5 nt 1682, or just upstream of the E1B coding segment. The PB-TREfragment was excised from CN250 with EagI and inserted into CN124digested with EagI. This produced CN254, which contains the PB-TREimmediately upstream of and operably linked to the E1B coding segment.

[0472] CN255 is identical to CN254, but the orientation of the PB-TREinsert is reversed.

[0473] CN275 is the same as CN254, but with a HindIII site at the5′-end.

[0474] PB-TRE-driven E1A and PB-TRE-driven E1B Ad5 plasmid (CN268)

[0475] An adenovirus vector in which expression of both E1A and E1B aredriven by PB-TRE was constructed as follows.

[0476] CN251, described above, comprises a PB-TRE fragment inserted justupstream of the E1A coding segment.

[0477] CN268 was generated by inserting a second PB-TRE in front of theE1B gene in CN251. A PB-TRE fragment was excised from CN250 byEagI-digestion and ligated into EagI-digested CN251 to create CN268. Thefinal construct is a plasmid with PB-TRE driving E1A and a second PB-TREdriving E1B. CN269 is the same as CN268 but the orientation of thesecond PB-TRE is reversed. Constructs CN251, CN254, and CN268 are shownschematically in FIG. 11.

EXAMPLE 7 Construction of Replication-Competent Adenoviral Vectors inWhich Adenoviral Genes are Under Transcriptional Control of a KallikreinTRE

[0478] hKLK2 promoter-driven E1A Ad5 plasmid CN303

[0479] CN303 was produced by inserting the minimal hKLK2 promoter (−324to +33) just upstream of the E1A coding segment in a derivative ofpXC-1, a plasmid containing the left hand end of the Ad5 genome.

[0480] CN124 is a derivative of construct pXC-1 which contains thewild-type left hand end of Ad5, including both E1A and E1B (McKinnon(1982) Gene 19:33-42). CN124 also has among other alterations, anartificial PinA1 site at Ad5 nt 547 (just upstream of the E1Atranscriptional start at nt 560 and the E1A coding segment beginningwith ATG at 610). CN124 was linearized with PinA1 and dephosphorylatedwith calf intestinal alkaline phosphatase (New England Biolab).

[0481] CN294 was digested with PinA1 to free the hKLK2 promoter. ThehKLK2 promoter was then ligated into the PinA1 linearized CN124,producing CN303. CN304 is similar to CN303 except for the hKLK2 promoterfragment is in the reverse orientation.

[0482] CN421 was constructed by inserting an hKLK2-TRE (comprising anhKLK2 enhancer from nucleotides −5155 to −3387 relative to the hKLK2gene transcription start site (nucleotides 6859 to 8627 of SEQ ID NO: 1)and an hKLK2 minimal promoter as in CN379; see Table 1 and FIG. 16) intoCN306. The hKLK2-TRE fragment was amplified by PCR from CN379, digestedwith PinA1 and ligated into similarly cut CN306, to produce CN421.

[0483] CN438 was constructed by inserting an hKLK2-TRE (comprising anhKLK2 enhancer from nucleotides −4814 to −3643 relative to the hKLK2gene transcription start site (nucleotides 7200 to 8371 of SEQ ID NO: 1)and a minimal hKLK2 promoter as in CN390; see Table 1 and FIG. 16) intoCN306. The enhancer fragment was amplified by PCR from CN390, digestedwith PinA1 and ligated into similarly cut CN306, to produce CN438.

[0484] CN306 was derived from CN124 by removing the endogenous64-nucleotide E1A promoter. CN124 is a derivative of construct pXC-1which contains the wild-type left hand end of Ad5, including both E1Aand E1B (McKinnon (1982) Gene 19:33-42). CN124 also has among otheralterations, an artificial PinA1 site at Ad5 nt 547 Oust upstream of theE1A transcriptional start at nt 560 and the E1A coding segment beginningwith ATG at 610). CN124 was linearized with PinA1 and dephosphorylatedwith calf intestinal alkaline phosphatase (New England Biolabs).

[0485] CN390 was constructed as follows. A fragment with KpnI and XhoIsites at the ends was amplified from CN379 with syntheticoligonucleotides 51.96.3 (5′-GAT CGG TAC CAA AAG CTT AGA GAT GAC CTCCC-3′; SEQ ID NO: 70) and 51.96.4 (5′-GAT CCT CGA GGC AAT AAT ACC GTTTTC TTT TCT GG-3′; SEQ ID NO: 71). The resulting fragment was digestedwith XhoI and KpnI, then cloned into similarly cut CN325, to generateCN390. CN390 has a 1.17-kb hKLK2 enhancer (nucleotides 7200 to 8371 ofSEQ ID NO: x)?? and a minimal hKLK2 promoter (−324 to +33 relative tothe transcription start site).

[0486] CN379 has, in addition to the minimal hKLK2 promoter, the hKLK25′ flanking region from −5155 to −3387 (nucleotides 6859 to 8627 of SEQID NO: 1) driving expression of the luciferase gene.

[0487] Construction of Adenovirus Vectors Comprising hKLK2-TREControlling Expression of Adenovirus E1A

[0488] CN749, comprising an hKLK2 promoter (−324 to +33) drivingadenovirus E1A gene expression, was generated by co-transfecting CN303and pBHG10 into 293 cells.

[0489] CN763, comprising an hKLK2-TRE promoter/enhancer from CN379controlling transcription of E1A, was generated from CN421 and pBHG10.To produce plasmid CN421, the hKLK2-TRE was amplified from CN379 andcloned into CN306. Therefore, CN763 is an adenoviral vector in which anhKLK2-TRE comprising a minimal hKLK2 promoter and the hKLK2 5′ flankingregion from −5155 to −3387 (nucleotides 6859 to 8627 of SEQ ID NO: 1)controls expression of E1A.

[0490] CN768, comprising an hKLK2-TRE controlling transcription of E1A,was constructed from CN438 and pBHG10. Thus, CN768 is an adenoviralvector in which an hKLK2-TRE (comprising an hKLK2 enhancer fromnucleotides −4814 to −3643 relative to the hKLK2 gene transcriptionstart site (nucleotides 7200 to 8371 of SEQ ID NO: 1) and a minimalhKLK2 promoter controls expression of E1A.

[0491] In Vitro Characterization of Adenoviral Constructs Comprising anAdenoviral Gene Under Transcriptional Control of an hKLK2-TRE

[0492] Plaque assays

[0493] To determine whether the adenoviral constructs described abovereplicate preferentially in prostate cells, plaque assays wereperformed. Plaquing efficiency was evaluated in the following celltypes: prostate tumor cell lines (LNCaP), breast normal cell line(HBL-100), ovarian tumor cell line (OVCAR-3, SK-OV-3), and human-embryonic kidney cells (293). LNCaP cells express both androgenreceptor and PSA, while the other cell lines tested do not. 293 cellsserve as a positive control for plaquing efficiency, since this cellline expresses Ad5 E1A and E1B proteins. The plaque assay was performedas follows: Confluent cell monolayers were seeded in 6-well disheseighteen hours before infection. The monolayers were infected with10-fold serial dilutions of each virus. After infecting monolayers forfour hours in serum-free media (MEM), the media was removed and replacedwith a solution of 0.75% low melting point agarose and tissue culturemedia. Plaques were scored two weeks after infection. CN702 has nomodifications in its E1 region and is used as a wild type control. CN706demonstrates selective cytotoxicity toward PSA-expressing cells in vitroand in vivo. Rodriguez et al. (1997) Cancer Res. 57:2559-2563. TABLE 12Viruses 293 LNCaP HBL-100 OVCAR-3 CN702 100 100  100 100 CN706 100 232.4 5.5 CN763 100 35 1.2 1.9 CN768 100 29 1.3 3.9

[0494] Table 12 shows the results of plaque assays performed with theadenoviral vectors described above. The results are expressed as percentof wild-type adenovirus plaque-forming units (PFU) per ml. The averagetiter of duplicate samples for the viruses tested. The titer for aparticular virus in all cell lines was normalized to its titer on 293cells. Once the titers on a cell type were normalized to 293 cells, thenormalized numbers of the recombinant viruses were compared to CN702. Aratio of less than 100 suggests that the virus tested plaques lessefficiently than CN702. Conversely, a ratio greater than 100 suggeststhat the virus plaques more efficiently than CN702.

[0495] The following observations were made. First, hKLK2-TRE engineeredadenoviruses demonstrate preferential replication in prostate tumorcells. Since this carcinoma expresses androgen receptors, the hKLK-TREcontained in the adenoviral vectors should be active in promoting thetranscription of the adenovirus early genes. The data presented in Table12 suggest that the hKLK2-TRE containing adenoviral vectors inducecytopathic effects with a lower efficiency than wild type adenovirus inprostate tumor cells. Second, hKLK2-TRE controlled adenoviruses show adramatically lower plaquing efficiency in non-prostate tumor cells whencompared to wild type. For example, in the ovarian carcinoma cell lineOVCAR-3, CN763 and CN768 produced about 25 to 50-fold less plaques thanwild type Ad5. The results are similar for these two viruses in HBL-100cells, where virus replication is also severely compromised. Third,PSA-TRE adenoviral vectors and hKLK2-TRE adenoviral vectors give similarplaques in HBL-100 and OVCAR-3 cells. Thus, like PSA-TRE adenoviralvector CN706, hKLK2-TRE adenoviral vectors were significantly attenuatedrelative to wild-type adenovirus in non-prostate cells, but thesevectors grew comparably in prostate tumor cells.

EXAMPLE 8 Preparation of Covalently Pegylated Adenovirus

[0496] A series of experiments were carried out to alter the surfacecapsids of adenovirus by complexing adenovirus with PEG. The objectiveof PEG complexation masking of the adenovirus surface is thefollowing: 1) adenovirus neutralizing antibodies or opsonins which arein circulation and 2) increase systemic circulation time of adenovirusparticles by reduction of non-specific clearance mechanisms in the body(i.e., macrophages, etc.) Surface capsids of wild type adenovirus CN706were modified through covalent attachment of PEG to hexon and fiberproteins using N-hydroxysuccinimidyl (NHS). The PEGs (Shearwaterpolymers, Inc.) had nominal molecular weight of 5000 Da. The activatedPEG (approximately 2 mM) was reacted with 5×10⁹ particles/ml adenovirusin a tris-HCI buffer (the approximate molar ratio of virus particle toPEG was 1:4×10⁶). Various combinations of pH, temperature, and reactiontime were used. After the reaction, unreacted activated PEG, unreactedadenovirus, and pegylated adenovirus were separated by anionic ionexchange chromatography on Q Sepharose XL (Pharmacia), running a 0 to1.5 M NaCl gradient in 50 mM tris, pH 8.0. The gradient was run over 10column volumes.

[0497] Characterization of PEG-CN706. Pegylation of CN706 was verifiedby SDS-page. FIG. 21 depicts the pegylation of CN706 and the mobilityshift of pegylated proteins. Lanes 1 and 2 are non-pegylated CN706(control), lanes 3 through 6 show the appearance of a second band abovethe hexon proteins of CN706, most likely pegylated hexon, and the lossof the fiber protein band. Since no additional bands associated with thevirus except that corresponding to the PEG-hexon protein, the pegylatedfiber protein is assumed to be under one of the unpegylated proteins onthe SDS gel.

[0498]FIG. 22 is an ion exchange chromatogram showing the change insurface properties of CN706. Pegylation of CN706 results from itsearlier elution from the Q sepharose resin used to capture the virus.This result is most likely due to PEG rendering the virus more chargeneutral in appearance and hence decreasing its binding-potential to theion exchange matrix. A broadening of the virus chromatogram is expectedsince the pegylation of CN706 occurs to different percentages.

[0499] The infectivity of pegylated CN706 was evaluated in an in vitroplaque assay on 293 cells. Table 13 depicts a 5-to 10-fold reduction inplaquing efficiency of PEG-CN706 and compared to CN706. This is mostlikely due to pegylation masking the virus cells, decreasing therecognition and endocytosis of the viral particles. TABLE 13 Comparisonof Plaquing Efficiency of CN706 and PEG-CN706 Sample Description Numberof Plaques (arbitrary units) CN706 15 ± 5 PEG-CN706  4 ± 1

[0500] It is evident from the above results that adenoviruses can beprovided as vehicles specific for particular host cells, where theviruses are replication-competent. The viruses may be vehicles for awide variety of genes for introduction in the target host cells.Particularly, employing the prostate specific element, the early genesessential for replication may be modified so as to be under the controlof prostate cell responsive elements. All publications and patentapplications mentioned in this specification are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The invention now being fully described, itwill be apparent to one of ordinary skill in the art that many changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

1 71 1 5836 DNA Homo sapiens 1 aagcttctag ttttcttttc ccggtgacatcgtggaaagc actagcatct ctaagcaatg 60 atctgtgaca atattcacag tgtaatgccatccagggaac tcaactgagc cttgatgtcc 120 agagattttt gtgttttttt ctgagactgagtctcgctct gtgccaggct ggagtgcagt 180 ggtgcaacct tggctcactg caagctccgcctcctgggtt cacgccattc tcctgcctca 240 gcctcctgag tagctgggac tacaggcacccgccaccacg cctggctaat ttttttgtat 300 ttttagtaga gatggggttt cactgtgttagccaggatgg tctcagtctc ctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgctgggatgacag gcgtgagcca ccgcgcctgg 420 ccgatatcca gagatttttt ggggggctccatcacacaga catgttgact gtcttcatgg 480 ttgactttta gtatccagcc cctctagaaatctagctgat atagtgtggc tcaaaacctt 540 cagcacaaat cacaccgtta gactatctggtgtggcccaa accttcaggt gaacaaaggg 600 actctaatct ggcaggatat tccaaagcattagagatgac ctcttgcaaa gaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaaaaaaaaaaaa gagatgacct ctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaaggtcagtcctc tgttgcacag tctccctcac 780 agggtcattg tgacgatcaa atgtggtcacgtgtatgagg caccagcaca tgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgcactgctgaatg cttgggatgt gtcagggatt 900 atcttcagca cttacagatg ctcatctcatcctcacagca tcactatggg atgggtatta 960 ctggcctcat ttgatggaga aagtggctgtggctcagaaa ggggggacca ctagaccagg 1020 gacactctgg atgctgggga ctccagagaccatgaccact caccaactgc agagaaatta 1080 attgtggcct gatgtccctg tcctggagagggtggaggtg gaccttcact aacctcctac 1140 cttgaccctc tcttttaggg ctctttctgacctccaccat ggtactagga ccccattgta 1200 ttctgtaccc tcttgactct atgacccccactgcccactg catccagctg ggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtctcagtgccca cctgtttgtc agtaactctg 1320 aaggggctga cattttactg acttgcaaacaaataagcta actttccaga gttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtcagaggcaaag gcttttactg ctcacagctt 1440 agcagacagc atgaggttca tgttcacattagtacacctt gcccccccca aatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgtgcagaagggg tttgtgccac tggtgagaaa 1560 cctgagatta ggaatcctca atcttatactgggacaactt gcaaacctgc tcagcctttg 1620 tctctgatga agatattatc ttcatgatcttggattgaaa acagacctac tctggaggaa 1680 catattgtat cgattgtcct tgacagtaaacaaatctgtt gtaagagaca ttatctttat 1740 tatctaggac agtaagcaag cctggatctgagagagatat catcttgcaa ggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccagaaaaaaaaag gtgttgctgt ctttgctcag 1860 aagacacaca gatacgtgac agaaccatggagaattgcct cccaacgctg ttcagccaga 1920 gccttccacc cttgtctgca ggacagtctcaacgttccac cattaaatac ttcttctatc 1980 acatcctgct tctttatgcc taaccaaggttctaggtccc gatcgactgt gtctggcagc 2040 actccactgc caaacccaga ataaggcagcgctcaggatc ccgaaggggc atggctgggg 2100 atcagaactt ctgggtttga gtgaggagtgggtccaccct cttgaatttc aaaggaggaa 2160 gaggctggat gtgaaggtac tgggggagggaaagtgtcag ttccgaactc ttaggtcaat 2220 gagggaggag actggtaagg tcccagctcccgaggtactg atgtgggaat ggcctaagaa 2280 tctcatatcc tcaggaagaa ggtgctggaatcctgagggg tagagttctg ggtatatttg 2340 tggcttaagg ctctttggcc cctgaaggcagaggctggaa ccattaggtc cagggtttgg 2400 ggtgatagta atgggatctc ttgattcctcaagagtctga ggatcgaggg ttgcccattc 2460 ttccatcttg ccacctaatc cttactccacttgagggtat caccagccct tctagctcca 2520 tgaaggtccc ctgggcaagc acaatctgagcatgaaagat gccccagagg ccttgggtgt 2580 catccactca tcatccagca tcacactctgagggtgtggc cagcaccatg acgtcatgtt 2640 gctgtgacta tccctgcagc gtgcctctccagccacctgc caaccgtaga gctgcccatc 2700 ctcctctggt gggagtggcc tgcatggtgccaggctgagg cctagtgtca gacagggagc 2760 ctggaatcat agggatccag gactcaaaagtgctagagaa tggccatatg tcaccatcca 2820 tgaaatctca agggcttctg ggtggagggcacagggacct gaacttatgg tttcccaagt 2880 ctattgctct cccaagtgag tctcccagatacgaggcact gtgccagcat cagccttatc 2940 tccaccacat cttgtaaaag gactacccagggccctgatg aacaccatgg tgtgtacagg 3000 agtagggggt ggaggcacgg actcctgtgaggtcacagcc aagggagcat catcatgggt 3060 ggggaggagg caatggacag gcttgagaacggggatgtgg ttgtatttgg ttttctttgg 3120 ttagataaag tgctgggtat aggattgagagtggagtatg aagaccagtt aggatggagg 3180 atcagattgg agttgggtta gataaagtgctgggtatagg attgagagtg gagtatgaag 3240 accagttagg atggaggatc agattggagttgggttagag atggggtaaa attgtgctcc 3300 ggatgagttt gggattgaca ctgtggaggtggtttgggat ggcatggctt tgggatggaa 3360 atagatttgt tttgatgttg gctcagacatccttggggat tgaactgggg atgaagctgg 3420 gtttgatttt ggaggtagaa gacgtggaagtagctgtcag atttgacagt ggccatgagt 3480 tttgtttgat ggggaatcaa acaatgggggaagacataag ggttggcttg ttaggttaag 3540 ttgcgttggg ttgatggggt cggggctgtgtataatgcag ttggattggt ttgtattaaa 3600 ttgggttggg tcaggttttg gttgaggatgagttgaggat atgcttgggg acaccggatc 3660 catgaggttc tcactggagt ggagacaaacttcctttcca ggatgaatcc agggaagcct 3720 taattcacgt gtaggggagg tcaggccactggctaagtat atccttccac tccagctcta 3780 agatggtctt aaattgtgat tatctatatccacttctgtc tccctcactg tgcttggagt 3840 ttacctgatc actcaactag aaacaggggaagattttatc aaattctttt tttttttttt 3900 ttttttttga gacagagtct cactctgttgcccaggctgg agtgcagtgg cgcagtctcg 3960 gctcactgca acctctgcct cccaggttcaagtgattctc ctgcctcagc ctcctgagtt 4020 gctgggatta caggcatgca gcaccatgcccagctaattt ttgtattttt agtagagatg 4080 gggtttcacc aatgtttgcc aggctggcctcgaactcctg acctggtgat ccacctgcct 4140 cagcctccca aagtgctggg attacaggcgtcagccaccg cgcccagcca cttttgtcaa 4200 attcttgaga cacagctcgg gctggatcaagtgagctact ctggttttat tgaacagctg 4260 aaataaccaa ctttttggaa attgatgaaatcttacggag ttaacagtgg aggtaccagg 4320 gctcttaaga gttcccgatt ctcttctgagactacaaatt gtgattttgc atgccacctt 4380 aatctttttt tttttttttt taaatcgaggtttcagtctc attctatttc ccaggctgga 4440 gttcaatagc gtgatcacag ctcactgtagccttgaactc ctggccttaa gagattctcc 4500 tgcttcggtc tcccaatagc taagactacagtagtccacc accatatcca gataattttt 4560 aaattttttg gggggccggg cacagtggctcacgcctgta atcccaacac catgggaggc 4620 tgagatgggt ggatcacgag gtcaggagtttgagaccagc ctgaccaaca tggtgaaact 4680 ctgtctctac taaaaaaaaa aaaaatagaaaaattagccg ggcgtggtgg cacacggcac 4740 ctgtaatccc agctactgag gaggctgaggcaggagaatc acttgaaccc agaaggcaga 4800 ggttgcaatg agccgagatt gcgccactgcactccagcct gggtgacaga gtgagactct 4860 gtctcaaaaa aaaaaaattt tttttttttttttgtagaga tggatcttgc tttgtttctc 4920 tggttggcct tgaactcctg gcttcaagtgatcctcctac cttggcctcg gaaagtgttg 4980 ggattacagg cgtgagccac catgactgacctgtcgttaa tcttgaggta cataaacctg 5040 gctcctaaag gctaaaggct aaatatttgttggagaaggg gcattggatt ttgcatgagg 5100 atgattctga cctgggaggg caggtcagcaggcatctctg ttgcacagat agagtgtaca 5160 ggtctggaga acaaggagtg gggggttattggaattccac attgtttgct gcacgttgga 5220 ttttgaaatg ctagggaact ttgggagactcatatttctg ggctagagga tctgtggacc 5280 acaagatctt tttatgatga cagtagcaatgtatctgtgg agctggattc tgggttggga 5340 gtgcaaggaa aagaatgtac taaatgccaagacatctatt tcaggagcat gaggaataaa 5400 agttctagtt tctggtctca gagtggtgcagggatcaggg agtctcacaa tctcctgagt 5460 gctggtgtct tagggcacac tgggtcttggagtgcaaagg atctaggcac gtgaggcttt 5520 gtatgaagaa tcggggatcg tacccaccccctgtttctgt ttcatcctgg gcatgtctcc 5580 tctgcctttg tcccctagat gaagtctccatgagctacaa gggcctggtg catccagggt 5640 gatctagtaa ttgcagaaca gcaagtgctagctctccctc cccttccaca gctctgggtg 5700 tgggaggggg ttgtccagcc tccagcagcatggggagggc cttggtcagc ctctgggtgc 5760 cagcagggca ggggcggagt cctggggaatgaaggtttta tagggctcct gggggaggct 5820 ccccagcccc aagctt 5836 2 5835 DNAHomo sapiens 2 aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatctctaagcaatg 60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagccttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggctggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattctcctgcctca 240 gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaatttttttgtat 300 ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctcctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagccaccgcgcctgg 420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgactgtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggctcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggtgaacaaaggg 600 actctaatct ggcaggatac tccaaagcat tagagatgac ctcttgcaaagaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacctctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacagtctccctcac 780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcacatgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgtgtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca tcactatgggatgggtatta 960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggaccactagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact caccaactgcagagaaatta 1080 attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcactaacctcctac 1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactaggaccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctgggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtcagtaactctg 1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccagagttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactgctcacagctt 1440 agcagacagc atgaggttca tgttcacatt agtacacctt gccccccccaaatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccactggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgctcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctactctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagacattatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaaggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttactgtctttgctcag 1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctgttcagccaga 1920 gccttccacc ctttctgcag gacagtctca acgttccacc attaaatacttcttctatca 1980 catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtgtctggcagca 2040 ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggcatggctgggga 2100 tcagaacttc tgggtttgag tgaggagtgg gtccaccctc ttgaatttcaaaggaggaag 2160 aggctggatg tgaaggtact gggggaggga aagtgtcagt tccgaactcttaggtcaatg 2220 agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatggcctaagaat 2280 ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgggtatatttgt 2340 ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtccagggtttggg 2400 gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggttgcccattct 2460 tccatcttgc cacctaatcc ttactccact tgagggtatc accagcccttctagctccat 2520 gaaggtcccc tgggcaagca caatctgagc atgaaagatg ccccagaggccttgggtgtc 2580 atccactcat catccagcat cacactctga gggtgtggcc agcaccatgacgtcatgttg 2640 ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagagctgcccatcc 2700 tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcagacagggagcc 2760 tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgtcaccatccat 2820 gaaatctcaa gggcttctgg gtggagggca cagggacctg aacttatggtttcccaagtc 2880 tattgctctc ccaagtgagt ctcccagata cgaggcactg tgccagcatcagccttatct 2940 ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggtgtgtacagga 3000 gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatcatcatgggtg 3060 gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggttttctttggt 3120 tagataaagt gctgggtata ggattgagag tggagtatga agaccagttaggatggagga 3180 tcagattgga gttgggttag ataaagtgct gggtatagga ttgagagtggagtatgaaga 3240 ccagttagga tggaggatca gattggagtt gggttagaga tggggtaaaattgtgctccg 3300 gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggctttgggatggaaa 3360 tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactggggatgaagctggg 3420 tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtggccatgagtt 3480 ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgttaggttaagt 3540 tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt tggattggtttgtattaaat 3600 tgggttgggt caggttttgg ttgaggatga gttgaggata tgcttggggacaccggatcc 3660 atgaggttct cactggagtg gagacaaact tcctttccag gatgaatccagggaagcctt 3720 aattcacgtg taggggaggt caggccactg gctaagtata tccttccactccagctctaa 3780 gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgtgcttggagtt 3840 tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttttttttttttt 3900 tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggcgcagtctcgg 3960 ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc tgcctcagcctcctgagttg 4020 ctgggattac aggcatgcag caccatgccc agctaatttt tgtatttttagtagagatgg 4080 ggtttcacca atgtttgcca ggctggcctc gaactcctga cctggtgatccacctgcctc 4140 agcctcccaa agtgctggga ttacaggcgt cagccaccgc gcccagccacttttgtcaaa 4200 ttcttgagac acagctcggg ctggatcaag tgagctactc tggttttattgaacagctga 4260 aataaccaac tttttggaaa ttgatgaaat cttacggagt taacagtggaggtaccaggg 4320 ctcttaagag ttcccgattc tcttctgaga ctacaaattg tgattttgcatgccacctta 4380 atcttttttt tttttttttt aaatcgaggt ttcagtctca ttctatttcccaggctggag 4440 ttcaatagcg tgatcacagc tcactgtagc cttgaactcc tggccttaagagattctcct 4500 gcttcggtct cccaatagct aagactacag tagtccacca ccatatccagataattttta 4560 aattttttgg ggggccgggc acagtggctc acgcctgtaa tcccaacaccatgggaggct 4620 gagatgggtg gatcacgagg tcaggagttt gagaccagcc tgaccaacatggtgaaactc 4680 tgtctctact aaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggcacacggcacc 4740 tgtaatccca gctactgagg aggctgaggc aggagaatca cttgaacccagaaggcagag 4800 gttgcaatga gccgagattg cgccactgca ctccagcctg ggtgacagagtgagactctg 4860 tctcaaaaaa aaaaaatttt tttttttttt ttgtagagat ggatcttgctttgtttctct 4920 ggttggcctt gaactcctgg cttcaagtga tcctcctacc ttggcctcggaaagtgttgg 4980 gattacaggc gtgagccacc atgactgacc tgtcgttaat cttgaggtacataaacctgg 5040 ctcctaaagg ctaaaggcta aatatttgtt ggagaagggg cattggattttgcatgagga 5100 tgattctgac ctgggagggc aggtcagcag gcatctctgt tgcacagatagagtgtacag 5160 gtctggagaa caaggagtgg ggggttattg gaattccaca ttgtttgctgcacgttggat 5220 tttgaaatgc tagggaactt tgggagactc atatttctgg gctagaggatctgtggacca 5280 caagatcttt ttatgatgac agtagcaatg tatctgtgga gctggattctgggttgggag 5340 tgcaaggaaa agaatgtact aaatgccaag acatctattt caggagcatgaggaataaaa 5400 gttctagttt ctggtctcag agtggtgcat ggatcaggga gtctcacaatctcctgagtg 5460 ctggtgtctt agggcacact gggtcttgga gtgcaaagga tctaggcacgtgaggctttg 5520 tatgaagaat cggggatcgt acccaccccc tgtttctgtt tcatcctgggcatgtctcct 5580 ctgcctttgt cccctagatg aagtctccat gagctacaag ggcctggtgcatccagggtg 5640 atctagtaat tgcagaacag caagtgctag ctctccctcc ccttccacagctctgggtgt 5700 gggagggggt tgtccagcct ccagcagcat ggggagggcc ttggtcagcctctgggtgcc 5760 agcagggcag gggcggagtc ctggggaatg aaggttttat agggctcctgggggaggctc 5820 cccagcccca agctt 5835 3 12047 DNA Homo sapiens 3gaattcagaa ataggggaag gttgaggaag gacactgaac tcaaagggga tacagtgatt 60ggtttatttg tcttctcttc acaacattgg tgctggagga attcccaccc tgaggttatg 120aagatgtctg aacacccaac acatagcact ggagatatga gctcgacaag agtttctcag 180ccacagagat tcacagccta gggcaggagg acactgtacg ccaggcagaa tgacatggga 240attgcgctca cgattggctt gaagaagcaa ggactgtggg aggtgggctt tgtagtaaca 300agagggcagg gtgaactctg attcccatgg gggaatgtga tggtcctgtt acaaattttt 360caagctggca gggaataaaa cccattacgg tgaggacctg tggagggcgg ctgccccaac 420tgataaagga aatagccagg tgggggcctt tcccattgta ggggggacat atctggcaat 480agaagccttt gagacccttt agggtacaag tactgaggca gcaaataaaa tgaaatctta 540tttttcaact ttatactgca tgggtgtgaa gatatatttg tttctgtaca gggggtgagg 600gaaaggaggg gaggaggaaa gttcctgcag gtctggtttg gtcttgtgat ccagggggtc 660ttggaactat ttaaattaaa ttaaattaaa acaagcgact gttttaaatt aaattaaatt 720aaattaaatt ttactttatt ttatcttaag ttctgggcta catgtgcagg acgtgcagct 780ttgttacata ggtaaacgtg tgccatggtg gtttgctgta cctatcaacc catcacctag 840gtattaagcc cagcatgcat tagctgtttt tcctgacgct ctccctctcc ctgactccca 900caacaggccc cagtgtgtgt tgttcccctc cctgtgtcca tgtgttctca ttgttcagct 960cccacttata agtgagaaca tgtggtgttt ggttttctgt ttctgtgtta gtttgctgag 1020gataatggct tccacctcca tccatgttcc tgcaaaggac gtgatcttat tcttttttat 1080ggttgcatag aaattgtttt tacaaatcca attgatattg tatttaatta caagttaatc 1140taattagcat actagaagag attacagaag atattaggta cattgaatga ggaaatatat 1200aaaataggac gaaggtgaaa tattaggtag gaaaagtata atagttgaaa gaagtaaaaa 1260aaaatatgca tgagtagcag aatgtaaaag aggtgaagaa cgtaatagtg actttttaga 1320ccagattgaa ggacagagac agaaaaattt taaggaattg ctaaaccatg tgagtgttag 1380aagtacagtc aataacatta aagcctcagg aggagaaaag aataggaaag gaggaaatat 1440gtgaataaat agtagagaca tgtttgatgg attttaaaat atttgaaaga cctcacatca 1500aaggattcat accgtgccat tgaagaggaa gatggaaaag ccaagaagcc agatgaaagt 1560tagaaatatt attggcaaag cttaaatgtt aaaagtccta gagagaaagg atggcagaaa 1620tattggcggg aaagaatgca gaacctagaa tataaattca tcccaacagt ttggtagtgt 1680gcagctgtag ccttttctag ataatacact attgtcatac atcgcttaag cgagtgtaaa 1740atggtctcct cactttattt atttatatat ttatttagtt ttgagatgga gcctcgctct 1800gtctcctagg ctggagtgca atagtgcgat accactcact gcaacctctg cctcctctgt 1860tcaagtgatt ttcttacctc agcctcccga gtagctggga ttacaggtgc gtgccaccac 1920acccggctaa tttttgtatt ttttgtagag acggggtttt gccatgttgg ccaggctggt 1980cttgaactcc tgacatcagg tgatccacct gccttggcct cctaaagtgc tgggattaca 2040ggcatgagcc accgtgccca accactttat ttatttttta tttttatttt taaatttcag 2100cttctatttg aaatacaggg ggcacatata taggattgtt acatgggtat attgaactca 2160ggtagtgatc atactaccca acaggtaggt tttcaaccca ctccccctct tttcctcccc 2220attctagtag tgtgcagtgt ctattgttct catgtttatg tctatgtgtg ctccaggttt 2280agctcccacc tgtaagtgag aacgtgtggt atttgatttt ctgtccctgt gttaattcac 2340ttaggattat ggcttccagc tccattcata ttgctgtaaa ggatatgatt catttttcat 2400ggccatgcag tattccatat tgcgtataga tcacattttc tttctttttt ttttttgaga 2460cggagtcttg ctttgctgcc taggctggag tgcagtagca cgatctcggc tcactgcaag 2520cttcacctcc ggggttcacg tcattcttct gtctcagctt cccaagtagc tgggactaca 2580ggcgcccgcc accacgtccg gctaattttt ttgtgtgttt ttagtagaga tgggggtttc 2640actgtgttag ccaggatggt cttgatctcc tgaccttgtg gtccacctgc ctcggtctcc 2700caaagtgctg ggattacagg ggtgagccac tgcgcccggc ccatatatac cacattttct 2760ttaaccaatc caccattgat gggcaactag gtagattcca tggattccac agttttgcta 2820ttgtgtgcag tgtggcagta gacatatgaa tgaatgtgtc tttttggtat aatgatttgc 2880attcctttgg gtatacagtc attaatagga gtgctgggtt gaacggtggc tctgtttaaa 2940attctttgag aattttccaa actgtttgcc atagagagca aactaattta catttccacg 3000aacagtatat aagcattccc ttttctccac agctttgtca tcatggtttt tttttttctt 3060tattttaaaa aagaatatgt tgttgttttc ccagggtaca tgtgcaggat gtgcaggttt 3120gttacatagg tagtaaacgt gagccatggt ggtttgctgc acctgtcaac ccattacctg 3180ggtatgaagc cctgcctgca ttagctcttt tccctaatgc tctcactact gccccaccct 3240caccctgaca gggcaaacag acaacctaca gaatgggagg aaatttttgc aatctattca 3300tctgacaaag gtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360aataatagcc actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg 3480agaagtgtct cttcatgcct tttggccact ttaatgggat tattttttgc tttttagttt 3540aagttcctta tagattctgg atattagact tcttattgga tgcatagttt gtgaatactc 3600tcttccattc tgtaggttgt ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660atcttagttt aattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720acttagtcat aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt 3840ttgtatatgt gaaaggtcta ctctcatttt ctttccctct ttctttcttt ctttcttttc 3900tttctttctt tctttctttc tttctttctt tctttctttc tttctttttg tccttctttc 3960tttctttctt tctctttctt tctctctttc tttttttttt ttgatggagt attgctctgt 4020tgcccaggct gcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080caactgattc tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc caggctggtt 4200tgaaactcct gacctcaaac gatctgcctg ccttggcctc ccaaagtgct gggattacag 4260gtgtgagcca ctgtgcccag ccaagaatgt cattttctaa gaggtccaag aacctcaaga 4320tattttggga ccttgagaag agaggaattc atacaggtat tacaagcaca gcctaatggc 4380aaatctttgg catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt 4500gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta ttttgcaaat 4560aaattggtcc tgctatgatt tactcttggt aagaacaggg aaaatagaga aaaatttaga 4620ttgcatctga cctttttttc tgaattttta tatgtgccta caatttgagc taaatcctga 4680attattttct ggttgcaaaa actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740acatttatct ggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag 4860aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg gtgaatctcc 4920tcatatgcaa tccactgggc tcgatctgct tcaaattgct gatgcactgc tgctaaagct 4980atacatttaa aaccctcact aaaggatcag ggaccatcat ggaagaggag gaaacatgaa 5040attgtaagag ccagattcgg ggggtagagt gtggaggtca gagcaactcc accttgaata 5100agaaggtaaa gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta 5220atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg agtagatttt 5280cttctttgtg gtgttacagt gatagtgagt gagttctcgt gagatctggt catttaaaag 5340tgtgtggccc ctcccctccc tctcttggtc ctcctactgc catgtaagat acctgctcct 5400gctttgcctt ctaccataag taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460tgcttcctgt acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat 5580tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat cctgtcctta 5640ggtcaaacaa ccttgatggc atcgtacttc aattgtctta cacattcctt ctgaatgact 5700cctcccctat ggcatataag ccctgggtct tgggggataa tggcagaggg gtccaccatc 5760ttgtctggct gccacctgag acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820ttctttctaa gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg 5940atagatataa tccacataaa taaaagctct tggagggccc tcaataattg ttaagagtgt 6000aaatgtgtcc aaagatggaa aatgtttgag aactactgtc ccagagattt tcctgagttc 6060tagagtgtgg gaatatagaa cctggagctt ggcttcttca gcctagaatc aggagtatgg 6120ggctgaagtc tgaagcttgg cttcagcagt ttggggttgg cttccggagc acatatttga 6180catgttgcga ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag 6300tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg gcatttcaac 6360ttcatataca gtgggctatc ttttggtcca tgtttcaacc aaacaaccga ataaaccatt 6420agaacctttc cccacttccc tagctgcaat gttaaaccta ggatttctgt ttaataggtt 6480catatgaata atttcagcct gatccaactt tacattcctt ctaccgttat tctacaccca 6540ccttaaaaat gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat 6660aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg acatcaattg 6720agatttcaag ggagtcgcca agatcatcct caggttcagt gattgctggt agccctcata 6780taactcaatg aaagctgtta tgctcatggc tatggtttat tacagcaaaa gaatagagat 6840gaaaatctag caagggaaga gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900attcctgttg ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga 7020gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact tagcccctcc 7080agacgtctac agctgatagg ctgtaaccca acattgtcac cataaatcac attgttagac 7140tatccagtgt ggcccaagct cccgtgtaaa cacaggcact ctaaacaggc aggatatttc 7200aaaagcttag agatgacctc ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260agaatccttt accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg 7380ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc atctaatcct 7440cacaacatgg ctacagggtg ggcactacta gcctcatttg acagaggaaa ggactgtgga 7500taagaagggg gtgaccaata ggtcagagtc attctggatg caaggggctc cagaggacca 7560tgattagaca ttgtctgcag agaaattatg gctggatgtc tctgccccgg aaagggggat 7620gcactttcct tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc 7740cactcttccc agctccatct ccagctggcc aggtgtagcc acagtaccta actctttgca 7800gagaactata aatgtgtatc ctacagggga gaaaaaaaaa aagaactctg aaagagctga 7860cattttaccg acttgcaaac acataagcta acctgccagt tttgtgctgg tagaactcat 7920gagactcctg ggtcagaggc aaaagatttt attacccaca gctaaggagg cagcatgaac 7980tttgtgttca catttgttca ctttgccccc caattcatat gggatgatca gagcagttca 8040ggtggatgga cacaggggtt tgtggcaaag gtgagcaacc taggcttaga aatcctcaat 8100cttataagaa ggtactagca aacttgtcca gtctttgtat ctgacggaga tattatcttt 8160ataattgggt tgaaagcaga cctactctgg aggaacatat tgtatttatt gtcctgaaca 8220gtaaacaaat ctgctgtaaa atagacgtta actttattat ctaaggcagt aagcaaacct 8280agatctgaag gcgataccat cttgcaaggc tatctgctgt acaaatatgc ttgaaaagat 8340ggtccagaaa agaaaacggt attattgcct ttgctcagaa gacacacaga aacataagag 8400aaccatggaa aattgtctcc caacactgtt cacccagagc cttccactct tgtctgcagg 8460acagtcttaa catcccatca ttagtgtgtc taccacatct ggcttcaccg tgcctaacca 8520agatttctag gtccagttcc ccaccatgtt tggcagtgcc ccactgccaa ccccagaata 8580agggagtgct cagaattccg aggggacatg ggtggggatc agaacttctg ggcttgagtg 8640cagagggggc ccatactcct tggttccgaa ggaggaagag gctggaggtg aatgtccttg 8700gaggggagga atgtgggttc tgaactctta aatccccaag ggaggagact ggtaaggtcc 8760cagcttccga ggtactgacg tgggaatggc ctgagaggtc taagaatccc gtatcctcgg 8820gaaggagggg ctgaaattgt gaggggttga gttgcagggg tttgttagct tgagactcct 8880tggtgggtcc ctgggaagca aggactggaa ccattggctc cagggtttgg tgtgaaggta 8940atgggatctc ctgattctca aagggtcaga ggactgagag ttgcccatgc tttgatcttt 9000ccatctactc cttactccac ttgagggtaa tcacctactc ttctagttcc acaagagtgc 9060gcctgcgcga gtataatctg cacatgtgcc atgtcccgag gcctggggca tcatccactc 9120atcattcagc atctgcgcta tgcgggcgag gccggcgcca tgacgtcatg tagctgcgac 9180tatccctgca gcgcgcctct cccgtcacgt cccaaccatg gagctgtgga cgtgcgtccc 9240ctggtggatg tggcctgcgt ggtgccaggc cggggcctgg tgtccgataa agatcctaga 9300accacaggaa accaggactg aaaggtgcta gagaatggcc atatgtcgct gtccatgaaa 9360tctcaaggac ttctgggtgg agggcacagg agcctgaact tacgggtttg ccccagtcca 9420ctgtcctccc aagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480accacatctt gtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa 9540gagggggtga aggcatggac tcctgtgtgg tcagagccca gagggggcca tgacgggtgg 9600ggaggaggct gtggactggc tcgagaagtg ggatgtggtt gtgtttgatt tcctttggcc 9660agataaagtg ctggatatag cattgaaaac ggagtatgaa gaccagttag aatggagggt 9720caggttggag ttgagttaca gatggggtaa aattctgctt cggatgagtt tggggattgg 9780caatctaaag gtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840tggctgggaa gggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga 9900atacatggag ctggctattg catgcgagga tgtgcattag tttggtttga tctttaaata 9960aaggaggcta ttagggttgt cttgaattag attaagttgt gttgggttga tgggttgggc 10020ttgtgggtga tgtggttgga ttgggctgtg ttaaattggt ttgggtcagg ttttggttga 10080ggttatcatg gggatgagga tatgcttggg acatggattc aggtggttct cattcaagct 10140gaggcaaatt tcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200ggccactggc tgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt 10260ctgtctcctt cactgtactt ggaattgatc tggtcattca gctggaaatg ggggaagatt 10320ttgtcaaatt cttgagacac agctgggtct ggatcagcgt aagccttcct tctggtttta 10380ttgaacagat gaaatcacat tttttttttc aaaatcacag aaatcttata gagttaacag 10440tggactctta taataagagt taacaccagg actcttattc ttgattcttt tctgagacac 10500caaaatgaga tttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560acagtctggg tcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca 10620ctgaaccctt gacctcctgg acttaaggga tcctcctgct tcagcctcct gagtagatgg 10680ggctacaggt gcttgccacc acacctggct aattaaattt tttttttttt tttgtagaga 10740aagggtctca ctttgttgcc ctggctgatc ttgaacttct gacttcaagt gattcttcag 10800ccttggactc ccaaagcact gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860gcttaatctt ggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag 10920gcattggatt ttgcatgagg acaattctga cctaggaggg caggtcaaca ggaatccccg 10980ctgtacctgt acgttgtaca ggcatggaga atgaggagtg aggaggccgt accggaaccc 11040catattgttt agtggacatt ggattttgaa ataataggga acttggtctg ggagagtcat 11100atttctggat tggacaatat gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160ggggaaccat tttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220tctatttcag gaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga 11280ccaggaagtc tcacaatccg atcattctga tatttcaggg catattaggt ttggggtgca 11340aaggaagtac ttgggactta ggcacatgag actttgtatt gaaaatcaat gattggggct 11400ggccgtggtg ctcacgcctg taatctcatc actttgggag accgaagtgg gaggatggct 11460tgatctcaag agttggacac cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520attaaaaatt agctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580gacaggagaa tcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct 11640gcactccagc ctgggaaaca gagtgagact gtctcagaat ttttttaaaa aagaatcagt 11700gatcatccca acccctgttg ctgttcatcc tgagcctgcc ttctctggct ttgttcccta 11760gatcacatct ccatgatcca taggccctgc ccaatctgac ctcacaccgt gggaatgcct 11820ccagactgat ctagtatgtg tggaacagca agtgctggct ctccctcccc ttccacagct 11880ctgggtgtgg gagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940taggtgccaa cagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag 12000ggaggccccc cagccccaaa ctgcaccacc tggccgtgga caccggt 12047 4 454 DNA Homosapiens 4 aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccacagttcaggttc 60 aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacaccaacatctat 120 ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattccagttaagaaa 180 atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcccacaaataaa 240 aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaaagtactccaa 300 gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactgccaactggga 360 tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagcaggaagctact 420 ctgcaccttg tcagtgaggt ccagatacct acag 454 5 5224 DNA Homosapiens 5 gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttcaccccataggt 60 gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcataagaattaagg 120 aacagactat gggctggagg actttgagga tgtctgtctc ataacacttgggttgtatct 180 gttctatggg gcttgtttta agcttggcaa cttgcaacag ggttcactgactttctcccc 240 aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggcttgaatatct 300 gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgagagatgtgtcat 360 tcatttgtat caatgaatga atgaggacaa ttagtgtata aatccttagtacaacaatct 420 gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttctatttattta 480 tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagtctttgaattt 540 aagaagttaa tggtccagga ataattacat agcttacaaa tgactatgatataccatcaa 600 acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaaggtgagaggg 660 ctcttctcta gctagagact aatcagaaat acattcaggg ataattatttgaatagacct 720 taagggttgg gtacattttg ttcaagcatt gatggagaag gagagtgaatatttgaaaac 780 attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaatctcagaaaca 840 gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactgctctgaagaa 900 gtatatatct aatatttaac actaacatca tgctaataat gataataattactgtcattt 960 tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatatatcaaaataa 1020 acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacagattgaattat 1080 ttgcctgtca tacagctaat aattgaccat aagacaatta gatttaaattagttttgaat 1140 ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggcttcacagact 1200 tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctgtgaactctgt 1260 acttaggact aaactttgag caataacaca catagattga ggattgtttgctgttagcat 1320 acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgctgttcttcat 1380 ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctacaataactgtc 1440 tgcaagctta tgattcccaa atatctatct ctagcctcaa tcttgttccagaagataaaa 1500 agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacgtttcaacata 1560 caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagcacatagggtcc 1620 tcttgttcct taaaatctaa ttacttttag cccagtgctc atcccacctatggggagatg 1680 agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatagagccaggact 1740 gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactgaacatgtactt 1800 agttactaag tctttgactt tatctcattc ataccactca gctttatccaggccacttat 1860 ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtcttatccccttt 1920 tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcccttttgttgt 1980 ctataatagt cccagaagga gttataaact ccatttaaaa agtctttgagatgtggccct 2040 tgccaacttt gccaggaatt cccaatatct agtattttct actattaaactttgtgcctc 2100 ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttcccttaccggttggtt 2160 tttccaccac cttttacatt ttcctggaac actataccct ccctcttcatttggcccacc 2220 tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagccttatctgacc 2280 cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatccatcacacca 2340 tgttgtgatt actgatacta ttgtctgttt ctctgattag gcagtaagctcaacaagagc 2400 tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgcctggaatgca 2460 gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgctactttgtgca 2520 agacccggtt ctgaggcatt tatatttatt gatttattta attctcatttaaccatgaag 2580 gaggtactat cactatcctt attttatagt tgataaagat aaagcccagagaaatgaatt 2640 aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccagttccccaact 2700 ttacgtgatt aatactgtgc tatactgcct ctctgatcat atggcatggaatgcagacat 2760 ctgctccgta aggcagaata tggaaggaga ttggaggatg acacaaaaccagcataatat 2820 cagaggaaaa gtccaaacag gacctgaact gatagaaaag ttgttactcctggtgtagtc 2880 gcatcgacat cttgatgaac tggtggctga cacaacatac attggcttgatgtgtacata 2940 ttatttgtag ttgtgtgtgt atttttatat atatatttgt aatattgaaatagtcataat 3000 ttactaaagg cctaccattt gccaggcatt tttacatttg tcccctctaatcttttgatg 3060 agatgatcag attggattac ttggccttga agatgatata tctacatctatatctatatc 3120 tatatctata tctatatcta tatctatatc tatatctata tatgtatatcagaaaagctg 3180 aaatatgttt tgtaaagtta taaagatttc agactttata gaatctgggatttgccaaat 3240 gtaacccctt tctctacatt aaacccatgt tggaacaaat acatttattattcattcatc 3300 aaatgttgct gagtcctggc tatgaaccag acactgtgaa agcctttgggatattttgcc 3360 catgcttggg caagcttata tagtttgctt cataaaactc tatttcagttcttcataact 3420 aatacttcat gactattgct tttcaggtat tccttcataa caaatactttggctttcata 3480 tatttgagta aagtccccct tgaggaagag tagaagaact gcactttgtaaatactatcc 3540 tggaatccaa acggatagac aaggatggtg ctacctcttt ctggagagtacgtgagcaag 3600 gcctgttttg ttaacatgtt ccttaggaga caaaacttag gagagacacgcatagcagaa 3660 aatggacaaa aactaacaaa tgaatgggaa ttgtacttga ttagcattgaagaccttgtt 3720 tatactatga taaatgtttg tatttgctgg aagtgctact gacggtaaaccctttttgtt 3780 taaatgtgtg ccctagtagc ttgcagtatg atctattttt taagtactgtacttagctta 3840 tttaaaaatt ttatgtttaa aattgcatag tgctctttca ttgaagaagttttgagagag 3900 agatagaatt aaattcactt atcttaccat ctagagaaac ccaatgttaaaactttgttg 3960 tccattattt ctgtctttta ttcaacattt tttttagagg gtgggaggaatacagaggag 4020 gtacaatgat acacaaatga gagcactctc catgtattgt tttgtcctgtttttcagtta 4080 acaatatatt atgagcatat ttccatttca ttaaatattc ttccacaaagttattttgat 4140 ggctgtatat caccctactt tatgaatgta ccatattaat ttatttcctggtgtgggtta 4200 tttgatttta taatcttacc tttagaataa tgaaacacct gtgaagctttagaaaatact 4260 ggtgcctggg tctcaactcc acagattctg atttaactgg tctgggttacagactaggca 4320 ttgggaattc aaaaagttcc cccagtgatt ctaatgtgta gccaagatcgggaacccttg 4380 tagacaggga tgataggagg tgagccactc ttagcatcca tcatttagtattaacatcat 4440 catcttgagt tgctaagtga atgatgcacc tgacccactt tataaagacacatgtgcaaa 4500 taaaattatt ataggacttg gtttattagg gcttgtgctc taagttttctatgttaagcc 4560 atacatcgca tactaaatac tttaaaatgt accttattga catacatattaagtgaaaag 4620 tgtttctgag ctaaacaatg acagcataat tatcaagcaa tgataatttgaaatgaattt 4680 attattctgc aacttaggga caagtcatct ctctgaattt tttgtactttgagagtattt 4740 gttatatttg caagatgaag agtctgaatt ggtcagacaa tgtcttgtgtgcctggcata 4800 tgataggcat ttaatagttt taaagaatta atgtatttag atgaattgcataccaaatct 4860 gctgtctttt ctttatggct tcattaactt aatttgagag aaattaattattctgcaact 4920 tagggacaag tcatgtcttt gaatattctg tagtttgagg agaatatttgttatatttgc 4980 aaaataaaat aagtttgcaa gttttttttt tctgccccaa agagctctgtgtccttgaac 5040 ataaaataca aataaccgct atgctgttaa ttattggcaa atgtcccattttcaacctaa 5100 ggaaatacca taaagtaaca gatataccaa caaaaggtta ctagttaacaggcattgcct 5160 gaaaagagta taaaagaatt tcagcatgat tttccatatt gtgcttccaccactgccaat 5220 aaca 5224 6 822 DNA Homo sapiens 6 gcattgctgt gaactctgtacttaggacta aactttgagc aataacacac atagattgag 60 gattgtttgc tgttagcatacaaactctgg ttcaaagctc ctctttattg cttgtcttgg 120 aaaatttgct gttcttcatggtttctcttt tcactgctat ctatttttct caaccactca 180 catggctaca ataactgtctgcaagcttat gattcccaaa tatctatctc tagcctcaat 240 cttgttccag aagataaaaagtagtattca aatgcacatc aacgtctcca cttggagggc 300 ttaaagacgt ttcaacatacaaaccgggga gttttgcctg gaatgtttcc taaaatgtgt 360 cctgtagcac atagggtcctcttgttcctt aaaatctaat tacttttagc ccagtgctca 420 tcccacctat ggggagatgagagtgaaaag ggagcctgat taataattac actaagtcaa 480 taggcataga gccaggactgtttgggtaaa ctggtcactt tatcttaaac taaatatatc 540 caaaactgaa catgtacttagttactaagt ctttgacttt atctcattca taccactcag 600 ctttatccag gccacttatgagctctgtgt ccttgaacat aaaatacaaa taaccgctat 660 gctgttaatt attggcaaatgtcccatttt caacctaagg aaataccata aagtaacaga 720 tataccaaca aaaggttactagttaacagg cattgcctga aaagagtata aaagaatttc 780 agcatgattt tccatattgtgcttccacca ctgccaataa ca 822 7 472 DNA Homo sapiens 7 agccaccacccagtgagcct ttttctagcc cccagagcca cctctgtcac cttcctgttg 60 ggcatcatcccaccttccca gagccctgga gagcatgggg agacccggga ccctgctggg 120 tttctctgtcacaaaggaaa ataatccccc tggtgtgaca gacccaagga cagaacacag 180 cagaggtcagcactggggaa gacaggttgt cctcccaggg gatgggggtc catccacctt 240 gccgaaaagatttgtctgag gaactgaaaa tagaagggaa aaaagaggag ggacaaaaga 300 ggcagaaatgagaggggagg ggacagagga cacctgaata aagaccacac ccatgaccca 360 cgtgatgctgagaagtactc ctgccctagg aagagactca gggcagaggg aggaaggaca 420 gcagaccagacagtcacagc agccttgaca aaacgttcct ggaactcaag ca 472 8 858 DNA Homosapiens 8 cgagcggccc ctcagcttcg gcgcccagcc ccgcaaggct cccggtgaccactagagggc 60 gggaggagct cctggccagt ggtggagagt ggcaaggaag gaccctagggttcatcggag 120 cccaggttta ctcccttaag tggaaatttc ttcccccact cctccttggctttctccaag 180 gagggaaccc aggctgctgg aaagtccggc tggggcgggg actgtgggttcaggggagaa 240 cggggtgtgg aacgggacag ggagcggtta gaagggtggg gctattccgggaagtggtgg 300 ggggagggag cccaaaacta gcacctagtc cactcattat ccagccctcttatttctcgg 360 ccgctctgct tcagtggacc cggggagggc ggggaagtgg agtgggagacctaggggtgg 420 gcttcccgac cttgctgtac aggacctcga cctagctggc tttgttccccatccccacgt 480 tagttgttgc cctgaggcta aaactagagc ccaggggccc caagttccagactgcccctc 540 ccccctcccc cggagccagg gagtggttgg tgaaaggggg aggccagctggagaacaaac 600 gggtagtcag ggggttgagc gattagagcc cttgtaccct acccaggaatggttggggag 660 gaggaggaag aggtaggagg taggggaggg ggcggggttt tgtcacctgtcacctgctcg 720 ctgtgcctag ggcgggcggg cggggagtgg ggggaccggt ataaagcggtaggcgcctgt 780 gcccgctcca cctctcaagc agccagcgcc tgcctgaatc tgttctgccccctccccacc 840 catttcacca ccaccatg 858 9 454 DNA Homo sapiens 9aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccaca gttcaggttc 60aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacac caacatctat 120ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattcc agttaagaaa 180atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcc cacaaataaa 240aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaa agtactccaa 300gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactg ccaactggga 360tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagca ggaagctact 420ctgcaccttg tcagtgaggt ccagatacct acag 454 10 307 DNA Homo sapiens CDS(2)..(304) 10 g atg acc ggc tca acc atc gcg ccc aca acg gac tat cgc aacacc act 49 Met Thr Gly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg Asn ThrThr 1 5 10 15 gct acc gga cta aca tct gcc cta aat tta ccc caa gtt catgcc ttt 97 Ala Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro Gln Val His AlaPhe 20 25 30 gtc aat gac tgg gcg agc ttg gac atg tgg tgg ttt tcc ata gcgctt 145 Val Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu35 40 45 atg ttt gtt tgc ctt att att atg tgg ctt att tgt tgc cta aag cgc193 Met Phe Val Cys Leu Ile Ile Met Trp Leu Ile Cys Cys Leu Lys Arg 5055 60 aga cgc gcc aga ccc ccc atc tat agg cct atc att gtg ctc aac cca241 Arg Arg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro 6570 75 80 cac aat gaa aaa att cat aga ttg gac ggt ctg aaa cca tgt tct ctt289 His Asn Glu Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu 8590 95 ctt tta cag tat gat taa 307 Leu Leu Gln Tyr Asp 100 11 101 PRTHomo sapiens 11 Met Thr Gly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg AsnThr Thr 1 5 10 15 Ala Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro Gln ValHis Ala Phe 20 25 30 Val Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe SerIle Ala Leu 35 40 45 Met Phe Val Cys Leu Ile Ile Met Trp Leu Ile Cys CysLeu Lys Arg 50 55 60 Arg Arg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile ValLeu Asn Pro 65 70 75 80 His Asn Glu Lys Ile His Arg Leu Asp Gly Leu LysPro Cys Ser Leu 85 90 95 Leu Leu Gln Tyr Asp 100 12 25 DNA UnknownDescription of Unknown Organism unknown 12 ggacctcgag gtctccatga gctac25 13 23 DNA Unknown Description of Unknown Organism unknown 13agctcgagct tcgggatcct gag 23 14 19 DNA Unknown Description of UnknownOrganism unknown 14 tcgtcttcaa gaattctca 19 15 20 DNA UnknownDescription of Unknown Organism unknown 15 tttcagtcac cggtgtcgga 20 1620 DNA Unknown Description of Unknown Organism unknown 16 gcattctctagacacaggtg 20 17 22 DNA Unknown Description of Unknown Organism unknown17 tccgacaccg ggtgacctga aa 22 18 29 DNA Unknown Description of UnknownOrganism unknown 18 cattaaccgg tacctctaga aaatctagc 29 19 27 DNA UnknownDescription of Unknown Organism unknown 19 cattaaccgg taagcttggg gctgggg27 20 26 DNA Unknown Description of Unknown Organism unknown 20ccgctcgaga tcacactccg ccacac 26 21 24 DNA Unknown Description of UnknownOrganism unknown 21 ccgctcgagc actcttgagt gcca 24 22 156 DNA UnknownDescription of Unknown Organism unknown 22 tcgagggatg ttgtagtaaatttgggcgta accgagtaag atttggccat tttcgcggga 60 aaactgaata agactcttcgaaatctgaat aattttgtgt tactcatagc gcgtaatatt 120 tgtctagggc cgcggggactttgaccgttt acgtgg 156 23 156 DNA Unknown Description of Unknown Organismunknown 23 gatcccacgt aaacggtcaa agtccccgcg gccctagaca aatattacgcgctatgagta 60 acacaaaatt attcagattt cgaagagtct tattcagttt tcccgcgaaaatggccaaat 120 cttactcggt tacgcccaaa tttactacaa catccc 156 24 27 DNAUnknown Description of Unknown Organism unknown 24 ggaagatctg aaatctagctgatatag 27 25 24 DNA Unknown Description of Unknown Organism unknown 25ttctcgagaa gcttggggct gggg 24 26 39 DNA Unknown Description of UnknownOrganism unknown 26 gtcgacgtga aatctgaata attttgtgtt actcatagc 39 27 23DNA Unknown Description of Unknown Organism unknown 27 caccggcgcacaccaaaaac gtc 23 28 21 DNA Unknown Description of Unknown Organismunknown 28 gcccacggcc gcattatata c 21 29 21 DNA Unknown Description ofUnknown Organism unknown 29 gtatataatg cggccgtggg c 21 30 21 DNA UnknownDescription of Unknown Organism unknown 30 ccagaaaatc cagcaggtac c 21 3126 DNA Unknown Description of Unknown Organism unknown 31 taacggccgtctagaaatct agctga 26 32 23 DNA Unknown Description of Unknown Organismunknown 32 taacggccga agcttgggct ggg 23 33 20 DNA Unknown Description ofUnknown Organism unknown 33 taactcacgt tgtgcattgt 20 34 21 DNA UnknownDescription of Unknown Organism unknown 34 ggtgccgtgc tcgagtggtg t 21 3521 DNA Unknown Description of Unknown Organism unknown 35 acaccactcgagcacggcac c 21 36 24 DNA Unknown Description of Unknown Organismunknown 36 gctactattc gacagtttgt actg 24 37 27 DNA Unknown Descriptionof Unknown Organism unknown 37 gggtcgacgt acctctagaa atctagc 27 38 30DNA Unknown Description of Unknown Organism unknown 38 gtttgtgtattttagatcaa agatgctgca 30 39 26 DNA Unknown Description of UnknownOrganism unknown 39 gcatctttga tctaaaatac acaaac 26 40 30 DNA UnknownDescription of Unknown Organism unknown 40 taaaggagga gatctgcctaaaacactgca 30 41 25 DNA Unknown Description of Unknown Organism unknown41 gtgttttagg cagatctcct ccttt 25 42 43 DNA Unknown Description ofUnknown Organism unknown 42 gcaacccacc ggtgctaatc aagtatggca aaggagtaagcgc 43 43 26 DNA Unknown Description of Unknown Organism unknown 43tggccttgct agactgctcc ttcagc 26 44 822 DNA Unknown Description ofUnknown Organism unknown 44 gcattgctgt gaactctgta cttaggacta aactttgagcaataacacac atagattgag 60 gattgtttgc tgttagcata caaactctgg ttcaaagctcctctttattg cttgtcttgg 120 aaaatttgct gttcttcatg gtttctcttt tcactgctatctatttttct caaccactca 180 catggctaca ataactgtct gcaagcttat gattcccaaatatctatctc tagcctcaat 240 cttgttccag aagataaaaa gtagtattca aatgcacatcaacgtctcca cttggagggc 300 ttaaagacgt ttcaacatac aaaccgggga gttttgcctggaatgtttcc taaaatgtgt 360 cctgtagcac atagggtcct cttgttcctt aaaatctaattacttttagc ccagtgctca 420 tcccacctat ggggagatga gagtgaaaag ggagcctgattaataattac actaagtcaa 480 taggcataga gccaggactg tttgggtaaa ctggtcactttatcttaaac taaatatatc 540 caaaactgaa catgtactta gttactaagt ctttgactttatctcattca taccactcag 600 ctttatccag gccacttatg agctctgtgt ccttgaacataaaatacaaa taaccgctat 660 gctgttaatt attggcaaat gtcccatttt caacctaaggaaataccata aagtaacaga 720 tataccaaca aaaggttact agttaacagg cattgcctgaaaagagtata aaagaatttc 780 agcatgattt tccatattgt gcttccacca ctgccaataa ca822 45 5224 DNA Unknown Description of Unknown Organism unknown 45gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttca ccccataggt 60gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg 120aacagactat gggctggagg actttgagga tgtctgtctc ataacacttg ggttgtatct 180gttctatggg gcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct 300gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360tcatttgtat caatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttc tatttattta 480tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagt ctttgaattt 540aagaagttaa tggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg 660ctcttctcta gctagagact aatcagaaat acattcaggg ataattattt gaatagacct 720taagggttgg gtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca 840gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900gtatatatct aatatttaac actaacatca tgctaataat gataataatt actgtcattt 960tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa 1020acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080ttgcctgtca tacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact 1200tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260acttaggact aaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat 1380ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctac aataactgtc 1440tgcaagctta tgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata 1560caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620tcttgttcct taaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatag agccaggact 1740gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800agttactaag tctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt 1920tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcc cttttgttgt 1980ctataatagt cccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040tgccaacttt gccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc 2100ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160tttccaccac cttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc 2280cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatc catcacacca 2340tgttgtgatt actgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca 2460gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgct actttgtgca 2520agacccggtt ctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580gaggtactat cactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt 2640aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccag ttccccaact 2700ttacgtgatt aatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760ctgctccgta aggcagaata tggaaggaga ttggaggatg acacaaaacc agcataatat 2820cagaggaaaa gtccaaacag gacctgaact gatagaaaag ttgttactcc tggtgtagtc 2880gcatcgacat cttgatgaac tggtggctga cacaacatac attggcttga tgtgtacata 2940ttatttgtag ttgtgtgtgt atttttatat atatatttgt aatattgaaa tagtcataat 3000ttactaaagg cctaccattt gccaggcatt tttacatttg tcccctctaa tcttttgatg 3060agatgatcag attggattac ttggccttga agatgatata tctacatcta tatctatatc 3120tatatctata tctatatcta tatctatatc tatatctata tatgtatatc agaaaagctg 3180aaatatgttt tgtaaagtta taaagatttc agactttata gaatctggga tttgccaaat 3240gtaacccctt tctctacatt aaacccatgt tggaacaaat acatttatta ttcattcatc 3300aaatgttgct gagtcctggc tatgaaccag acactgtgaa agcctttggg atattttgcc 3360catgcttggg caagcttata tagtttgctt cataaaactc tatttcagtt cttcataact 3420aatacttcat gactattgct tttcaggtat tccttcataa caaatacttt ggctttcata 3480tatttgagta aagtccccct tgaggaagag tagaagaact gcactttgta aatactatcc 3540tggaatccaa acggatagac aaggatggtg ctacctcttt ctggagagta cgtgagcaag 3600gcctgttttg ttaacatgtt ccttaggaga caaaacttag gagagacacg catagcagaa 3660aatggacaaa aactaacaaa tgaatgggaa ttgtacttga ttagcattga agaccttgtt 3720tatactatga taaatgtttg tatttgctgg aagtgctact gacggtaaac cctttttgtt 3780taaatgtgtg ccctagtagc ttgcagtatg atctattttt taagtactgt acttagctta 3840tttaaaaatt ttatgtttaa aattgcatag tgctctttca ttgaagaagt tttgagagag 3900agatagaatt aaattcactt atcttaccat ctagagaaac ccaatgttaa aactttgttg 3960tccattattt ctgtctttta ttcaacattt tttttagagg gtgggaggaa tacagaggag 4020gtacaatgat acacaaatga gagcactctc catgtattgt tttgtcctgt ttttcagtta 4080acaatatatt atgagcatat ttccatttca ttaaatattc ttccacaaag ttattttgat 4140ggctgtatat caccctactt tatgaatgta ccatattaat ttatttcctg gtgtgggtta 4200tttgatttta taatcttacc tttagaataa tgaaacacct gtgaagcttt agaaaatact 4260ggtgcctggg tctcaactcc acagattctg atttaactgg tctgggttac agactaggca 4320ttgggaattc aaaaagttcc cccagtgatt ctaatgtgta gccaagatcg ggaacccttg 4380tagacaggga tgataggagg tgagccactc ttagcatcca tcatttagta ttaacatcat 4440catcttgagt tgctaagtga atgatgcacc tgacccactt tataaagaca catgtgcaaa 4500taaaattatt ataggacttg gtttattagg gcttgtgctc taagttttct atgttaagcc 4560atacatcgca tactaaatac tttaaaatgt accttattga catacatatt aagtgaaaag 4620tgtttctgag ctaaacaatg acagcataat tatcaagcaa tgataatttg aaatgaattt 4680attattctgc aacttaggga caagtcatct ctctgaattt tttgtacttt gagagtattt 4740gttatatttg caagatgaag agtctgaatt ggtcagacaa tgtcttgtgt gcctggcata 4800tgataggcat ttaatagttt taaagaatta atgtatttag atgaattgca taccaaatct 4860gctgtctttt ctttatggct tcattaactt aatttgagag aaattaatta ttctgcaact 4920tagggacaag tcatgtcttt gaatattctg tagtttgagg agaatatttg ttatatttgc 4980aaaataaaat aagtttgcaa gttttttttt tctgccccaa agagctctgt gtccttgaac 5040ataaaataca aataaccgct atgctgttaa ttattggcaa atgtcccatt ttcaacctaa 5100ggaaatacca taaagtaaca gatataccaa caaaaggtta ctagttaaca ggcattgcct 5160gaaaagagta taaaagaatt tcagcatgat tttccatatt gtgcttccac cactgccaat 5220aaca 5224 46 21 DNA Unknown Description of Unknown Organism Unknown 46gcccacggcc gcattatata c 21 47 21 DNA Unknown Description of UnknownOrganism Unknown 47 gtatataatg cggccgtggg c 21 48 29 DNA UnknownDescription of Unknown Organism Unknown 48 gtgaccggtg cattgctgtgaactctgta 29 49 27 DNA Unknown Description of Unknown Organism Unknown49 ataagtggcc tggataaagc tgagtgg 27 50 28 DNA Unknown Description ofUnknown Organism Unknown 50 gtcaccggtc tttgttattg gcagtggt 28 51 30 DNAUnknown Description of Unknown Organism Unknown 51 atccaggcca cttatgagctctgtgtcctt 30 52 26 DNA Unknown Description of Unknown Organism Unknown52 tatcggccgg cattgctgtg aactct 26 53 26 DNA Unknown Description ofUnknown Organism Unknown 53 ttacggccgc tttgttattg gcagtg 26 54 472 DNAUnknown Description of Unknown Organism Unknown 54 agccaccacc cagtgagcctttttctagcc cccagagcca cctctgtcac cttcctgttg 60 ggcatcatcc caccttcccagagccctgga gagcatgggg agacccggga ccctgctggg 120 tttctctgtc acaaaggaaaataatccccc tggtgtgaca gacccaagga cagaacacag 180 cagaggtcag cactggggaagacaggttgt cctcccaggg gatgggggtc catccacctt 240 gccgaaaaga tttgtctgaggaactgaaaa tagaagggaa aaaagaggag ggacaaaaga 300 ggcagaaatg agaggggaggggacagagga cacctgaata aagaccacac ccatgaccca 360 cgtgatgctg agaagtactcctgccctagg aagagactca gggcagaggg aggaaggaca 420 gcagaccaga cagtcacagcagccttgaca aaacgttcct ggaactcaag ca 472 55 26 DNA Unknown Description ofUnknown Organism Unknown 55 attaccggta gccaccaccc agtgag 26 56 26 DNAUnknown Description of Unknown Organism Unknown 56 tagaccggtg cttgagttccaggaac 26 57 21 DNA Unknown Description of Unknown Organism Unknown 57atttgtctag ggccgggact t 21 58 24 DNA Unknown Description of UnknownOrganism Unknown 58 cgcgcgcaaa acccctaaat aaag 24 59 21 DNA UnknownDescription of Unknown Organism Unknown 59 taacggccga gccaccaccc a 21 6023 DNA Unknown Description of Unknown Organism Unknown 60 tatcggccggcttgagttcc agg 23 61 307 DNA Unknown Description of Unknown OrganismUnknown 61 gatgaccggc tcaaccatcg cgcccacaac ggactatcgc aacaccactgctaccggact 60 aacatctgcc ctaaatttac cccaagttca tgcctttgtc aatgactgggcgagcttgga 120 catgtggtgg ttttccatag cgcttatgtt tgtttgcctt attattatgtggcttatttg 180 ttgcctaaag cgcagacgcg ccagaccccc catctatagg cctatcattgtgctcaaccc 240 acacaatgaa aaaattcata gattggacgg tctgaaacca tgttctcttcttttacagta 300 tgattaa 307 62 26 DNA Unknown Description of UnknownOrganism Unknown 62 taatccggac ggtgaccact agaggg 26 63 26 DNA UnknownDescription of Unknown Organism Unknown 63 tattccggat cacttaggca gcgctg26 64 24 DNA Unknown Description of Unknown Organism Unknown 64taacggccgc ggtgaccact agag 24 65 24 DNA Unknown Description of UnknownOrganism Unknown 65 tatcggccgg cagaacagat tcag 24 66 34 DNA UnknownDescription of Unknown Organism Unknown 66 gatcaccggt aagcttccacaagtgcattt agcc 34 67 33 DNA Unknown Description of Unknown OrganismUnknown 67 gatcaccggt ctgtaggtat ctggacctca ctg 33 68 34 DNA UnknownDescription of Unknown Organism Unknown 68 gatccggccg aagcttccacaagtgcattt agcc 34 69 33 DNA Unknown Description of Unknown OrganismUnknown 69 gatccggccg ctgtaggtat ctggacctca ctg 33 70 32 DNA UnknownDescription of Unknown Organism Unknown 70 gatcggtacc aaaagcttagagatgacctc cc 32 71 35 DNA Unknown Description of Unknown OrganismUnknown 71 gatcctcgag gcaataatac cgttttcttt tctgg 35

What is claimed is:
 1. A composition comprising: a replication competentadenovirus having an adenovirus gene essential for replication undertranscriptional control of a cell type specific transcriptional responseelement (TRE); and a masking agent, wherein the masking agent ispolyethylene glycol (PEG).
 2. The composition according to claim 1,wherein said adenovirus gene is an early gene.
 3. The compositionaccording to claim 2, wherein said early gene is selected from the groupconsisting of E1A, E1B and E4.
 4. The composition according to claim 1,wherein said adenovirus further comprises a transgene.
 5. Thecomposition according to claim 1, wherein said cell type specific TRE isprostate cell specific.
 6. The composition according to claim 5, whereinsaid adenovirus is replication-competent only in mammalian cellsexpressing prostate specific antigen.
 7. The composition according toclaim 5, wherein said promoter comprises nucleotides −540 to +8 relativeto transcription start site of prostate specific antigen gene.
 8. Thecomposition according to claim 5, wherein said prostate cell specificresponse element comprises the enhancer sequence in the region −5322 to−3739 relative to the transcription start site of the prostate specificantigen gene.
 9. The composition according to claim 5, wherein saidprostate cell specific response element is a probasin TRE.
 10. Thecomposition according to claim 5, wherein said prostate cell specificresponse element is a hKLK2-TRE.
 11. The composition according to claim1, wherein said adenovirus gene essential for replication is undertranscriptional control of an α-fetoprotein transcriptional regulatoryelement (AFP-TRE).
 12. The composition according to claim 1, whereinsaid adenovirus gene essential for replication is under transcriptionalcontrol of a MUC1-TRE.
 13. The composition according to claim 1, whereinsaid adenovirus gene essential for replication is under transcriptionalcontrol of a CEA-TRE.
 14. The composition according to claim 1, furthercomprising a physiologically acceptable carrier.
 15. The compositionaccording to claim 1, wherein said PEG is of a molecular weight betweenabout 2500 to about 30,000.
 16. The composition according to claim 15,wherein said PEG is of a molecular weight between about 3000 to about20,000.
 17. The composition of claim 16, wherein said PEG is of amolecular weight between about 5000 to about 10,000.
 18. The compositionaccording to claim 1, wherein said PEG is covalently attached to saidadenovirus.
 19. The composition according to claim 1, wherein said PEGis non-covalently attached to said adenovirus.
 20. The compositionaccording to claim 18, wherein the PEG is covalently attached to theadenovirus by using N-hydroxysuccinimidyl (NHS) active ester.
 21. Thecomposition according to claim 20, wherein said N-hydroxysuccinimidyl(NHS) active ester is selected from the group consisting of succinimidylsuccinate, succinimidyl succinamide, and succinimidyl propionate. 22.The composition according to claim 21, wherein saidN-hydroxysuccinimidyl (NHS) active ester is succinimidyl succinate. 23.An adenovirus, wherein proteins present in said adenovirus arecovalently attached to polyethylene glycol.
 24. The compositionaccording to claim 23, wherein said PEG is of a molecular weight betweenabout 2500 to about 30,000.
 25. The composition according to claim 24,wherein said PEG is of a molecular weight between about 3000 to about20,000.
 26. The composition of claim 25, wherein said PEG is of amolecular weight between about 5000 to about 10,000.
 27. The compositionaccording to claim 23, wherein the PEG is covalently attached to theadenovirus by using N-hydroxysuccinimidyl (NHS) active ester.
 28. Thecomposition according to claim 27, wherein said N-hydroxysuccinimidyl(NHS) active ester is selected from the group consisting of succinimidylsuccinate, succinimidyl succinamide, and succinimidyl propionate.